Glycosylation-resistant cyanovirins and related conjugates, compositions, nucleic acids, vectors, host cells, methods of production and methods of using nonglycosylated cyanovirins

ABSTRACT

The invention provides a method of inhibiting prophylactically or therapeutically an influenza viral infection in a host. The method comprises instilling into or onto a host a cell producing an antiviral protein, antiviral peptide, or antiviral conjugate comprising at least nine contiguous amino acids of SEQ ID NO: 2, wherein the at least nine contiguous amino acids are nonglycosylated and have antiviral activity, whereupon the influenza viral infection is inhibited.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application is a continuation of co-pending patentapplication no. 09/815,079, which was filed on Mar. 22, 2001.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to the use of cyanovirins toinhibit prophylactically or therapeutically influenza viral infection,as well as glycosylation-resistant cyanovirins and related conjugates,compositions, nucleic acids, vectors, host cells and methods ofproduction and use.

BACKGROUND OF THE INVENTION

[0003] The field of viral therapeutics has developed in response to theneed for agents effective against retroviruses, especially HIV. Thereare many ways in which an agent can exhibit anti-retroviral activity(e.g., see DeClercq, Adv. Virus Res. 42, 1-55, 1993; DeClercq, J.Acquir. Immun. Def. Synd. 4, 207-218, 1991; and Mitsuya et al., Science249, 1533-1544, 1990). Nucleoside derivatives, such as AZT, whichinhibit the viral reverse transcriptase, were among the first clinicallyactive agents available commercially for anti-HIV therapy. Although veryuseful in some patients, the utility of AZT and related compounds islimited by toxicity and insufficient therapeutic indices for fullyadequate therapy. Also, given the subsequent revelations about the truedynamics of HIV infection (Coffin, Science 267, 483-489, 1995; andCohen, Science 267, 179, 1995), it has become increasingly apparent thatagents acting as early as possible in the viral replicative cycle areneeded to inhibit infection of newly produced, uninfected immune cellsgenerated in the body in response to the virus-induced killing ofinfected cells. Also, it is essential to neutralize or inhibit newinfectious virus produced by infected cells.

[0004] Infection of people by influenza viruses is also a major cause ofpandemic illness, morbidity and mortality worldwide. The adverseeconomic consequences, as well as human suffering, are enormous.Available treatments for established infection by this virus are eitherminimally effective or ineffective; these treatments employamantatadine, rimantadine and neuraminidase inhibitors. Of these drugs,only the neuraminidase inhibitors are substantially active againstmultiple strains of influenza virus that commonly infect humans, yetthese drugs still have limited utility or efficacy against pandemicdisease.

[0005] Currently, the only effective preventative treatment againstinfluenza viral infection is vaccination. However, this, like the drugtreatments, is severely limited by the propensity of influenza virusesto mutate rapidly by genetic exchange, resulting in the emergence ofhighly resistant viral strains that rapidly infect and spread throughoutsusceptible populations. In fact, a vaccination strategy is onlyeffective from year-to-year if the potential pandemic strains can beidentified or predicted, and corresponding vaccines prepared andadministered early enough that the year's potential pandemic can beaborted or attenuated. Thus, new preventative and therapeuticinterventions and agents are urgently needed to combat influenzaviruses.

[0006] New agents with broad anti-influenza virus activity againstdiverse strains, clinical isolates and subtypes of influenza virus wouldbe highly useful, since such agents would most likely remain activeagainst the mutating virus. The two major types of influenza virus thatinfect humans are influenza A and B, both of which cause severe acuteillness that may include both respiratory and gastrointestinal distress,as well as other serious pathological sequellae. An agent that hasanti-influenza virus activity against diverse strains and isolates ofboth influenza A and B, including recent clinical isolates thereof,would be particularly advantageous for use in prevention or treatment ofhosts susceptible to influenza virus infection.

[0007] The predominant mode of transmission of influenza viral infectionis respiratory, i.e., transmission via inhalation of virus-ladenaerosolized particles generated through coughing, sneezing, breathing,etc., of an influenza-infected individual. Transmission of infectiousinfluenza virions may also occur through contact (e.g., throughinadvertent hand-to-mouth contact, kissing, touching, etc.) with salivaor other bodily secretions of an infected individual. Thus, the primaryfirst points of contact of infectious influenza virions within asusceptible individual are the mucosal surfaces within the oropharyngealmucosa, and the mucosal surfaces within the upper and lower respiratorytracts. Not only do these sites comprise first points of virus contactfor initial infection of an individual, they are also the primary sitesfor production and exit (e.g., by coughing, sneezing, salivarytransmission, etc.) of bodily fluids containing infectious influenzaviral particles. Therefore, availability of a highly potentanti-influenza virus agent, having broad-spectrum activity againstdiverse strains and isolates of influenza viruses A and B, which couldbe applied or delivered topically to the aforementioned mucosal sites ofcontact and infection and transmission of infectious influenza viruses,would be highly advantageous for therapeutic and preventative inhibitionof influenza viral infection, either in susceptible uninfected orinfected hosts.

[0008] In this regard, new classes of antiviral agents, to be used aloneor in combination existing antiviral agents, are needed for effectiveantiviral therapy. New agents are also important for the prophylacticinhibition of viral infection. In both areas of need, the ideal newagent(s) would act as early as possible in the viral life cycle; be asvirus-specific as possible (i.e., attack a molecular target specific tothe virus but not the host); render the intact virus noninfectious;prevent the death or dysfunction of virus-infected cells; preventfurther production of virus from infected cells; prevent spread of virusinfection to uninfected cells; be highly potent and active against thebroadest possible range of strains and isolates of a given virus; beresistant to degradation under physiological and rigorous environmentalconditions; and be readily and inexpensively produced.

[0009] Accordingly, it is an object of the present invention to providea method of inhibiting prophylactically or therapeutically a viralinfection, specifically an influenza viral infection, of a host. It isanother object of the present invention to provideglycosylation-resistant cyanovirins and related conjugates, nucleicacids, vectors, host cells and methods of production and use. These andother objects and advantages of the present invention, as well asadditional inventive features, will become apparent from the descriptionprovided herein.

BRIEF SUMMARY OF THE INVENTION

[0010] The present invention provides, among other things, an isolatedand purified nucleic acid molecule that encodes a protein or peptidecomprising at least nine contiguous amino acids of SEQ ID NO:2, whereinthe at least nine contiguous amino acids comprise amino acids 30-32 ofSEQ ID NO: 2 which have been rendered glycosylation resistant andwherein the at least nine contiguous amino acids have antiviralactivity. Further provided is a vector comprising such an isolated andpurified nucleic acid molecule and a host cell or organism comprisingthe vector.

[0011] Accordingly, the present invention also provides a method ofproducing an antiviral protein or antiviral peptide, which methodcomprises expressing the vector in a host cell or organism. Thus, anantiviral protein or antiviral peptide comprising at least ninecontiguous amino acids of SEQ ID NO:2, wherein the at least ninecontiguous amino acids comprise amino acids 30-32 of SEQ ID NO: 2 whichhave been rendered glycosylation resistant and wherein the at least ninecontiguous amino acids have antiviral activity, is also provided as is aconjugate comprising the antiviral protein or antiviral peptide and atleast one effector component selected from the group consisting ofpolyethylene glycol, albumin, dextran, a toxin and an immunologicalreagent. Compositions comprising an effective amount of the antiviralprotein, antiviral peptide, antiviral protein conjugate or antiviralpeptide conjugate are also provided.

[0012] The present invention further provides a method of inhibitingprophylactically or therapeutically a viral infection, specifically aninfluenza viral infection, of a host. The method comprises administeringto the host an effective amount of an antiviral protein, antiviralpeptide, antiviral protein conjugate or antiviral peptide conjugatecomprising at least nine contiguous amino acids of SEQ ID NO:2, whereinthe at least nine contiguous amino acids are nonglycosylated and haveantiviral activity, whereupon the viral infection is inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1A is a graph of OD (206 nm) versus time (min), whichrepresents an HPLC chromatogram of nonreduced cyanovirin.

[0014]FIG. 1B is a bar graph of maximum dilution for 50% protectionversus HPLC fraction, which illustrates the maximum dilution of eachHPLC fraction that provided 50% protection from the cytopathic effectsof HIV infection for the nonreduced cyanovirin HPLC fractions.

[0015]FIG. 1C is a graph of OD (206) nm versus time (min), whichrepresents an HPLC chromatogram of reduced cyanovirin.

[0016]FIG. 1D is a bar graph of maximum dilution for 50% protectionversus HPLC dilution, which illustrates the maximum dilution of eachfraction that provided 50% protection from the cytopathic effects of HIVinfection for the reduced cyanovirin HPLC fractions.

[0017]FIG. 2 shows an example of a DNA sequence encoding a syntheticcyanovirin gene (SEQ ID NOS: 1-4).

[0018]FIG. 3 illustrates a site-directed mutagenesis maneuver used toeliminate codons for a FLAG octapeptide and a Hind III restriction sitefrom the sequence of FIG. 2.

[0019]FIG. 4 shows a typical HPLC chromatogram during the purificationof a recombinant native cyanovirin.

[0020]FIG. 5A is a graph of % control versus concentration (nM), whichillustrates the antiviral activity of native cyanovirin from Nostocellipsosporum.

[0021]FIG. 5B is a graph of % control versus concentration (nM), whichillustrates the antiviral activity of recombinant cyanovirin.

[0022]FIG. 5C is a graph of % control versus concentration (nM), whichillustrates the antiviral activity of recombinant FLAG-fusioncyanovirin.

[0023]FIG. 6A is a graph of % control versus concentration (nM), whichdepicts the relative numbers of viable CEM-SS cells infected with HIV-1in a BCECF assay.

[0024]FIG. 6B is a graph of % control versus concentration (nM), whichdepicts the relative DNA contents of CEM-SS cell cultures infected withHIV-1.

[0025]FIG. 6C is a graph of % control versus concentration (nM), whichdepicts the relative numbers of viable CEM-SS cells infected with HIV-1in an XTT assay.

[0026]FIG. 6D is a graph of % control versus concentration (nM), whichdepicts the effect of a range of concentration of cyanovirin uponindices of infectious virus or viral replication.

[0027]FIG. 7 is a graph of % uninfected control versus time of addition(hrs), which shows results of time-of-addition studies of a cyanovirin,showing anti-HIV activity in CEM-SS cells infected with HIV-1_(RF).

[0028]FIG. 8 is a graph of OD (450 nm) versus cyanovirin concentration(μg/ml), which illustrates cyanovirin/gp120 interactions defining gp120as a principal molecular target of cyanovirin.

[0029]FIG. 9 schematically illustrates a DNA coding sequence comprisinga FLAG-cyanovirin-N coding sequence coupled to a Pseudomonas exotoxincoding sequence.

[0030]FIG. 10 is a graph of OD (450 nM) versus PPE concentration (nM),which illustrates selective killing of viral gp 120-expressing (H9/IIIB)cells by a FLAG-cyanovirin-N/Pseudomonas exotoxin protein conjugate(PPE).

[0031]FIG. 11 is a flowchart of the synthesis of a cyanovirin gene.

[0032]FIG. 12 is a flowchart of the expression of synthetic cyanovirin.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

[0033] The principal overall objective of the present invention is toprovide anti-viral proteins, peptides and derivatives thereof, and broadmedical uses thereof, including prophylactic and/or therapeuticapplications against viruses. An initial observation, which led to thepresent invention, was antiviral activity in certain extracts fromcultured cyanobacteria (blue-green algae) tested in an anti-HIV screen.The screen is one that was conceived in 1986 (by M. R. Boyd of theNational Institutes of Health) and has been developed and operated atthe U.S. National Cancer Institute (NCI) since 1988 (see Boyd, in AIDS,Etiology Diagnosis. Treatment and Prevention, DeVita et al., eds.,Philadelphia: Lippincott, 1988, pp. 305-317).

[0034] Cyanobacteria (blue-green algae) were specifically chosen foranti-HIV screening because they had been known to produce a wide varietyof structurally unique and biologically active non-nitrogenous and aminoacid-derived natural products (Faulkner, Nat. Prod. Rep. 11, 355-394,1994; and Glombitza et al., in Algal and Cyanobacterial Biotechnology,Cresswell, R. C., et al. eds., 1989, pp. 211-218). These photosyntheticprocaryotic organisms are significant producers of cyclic and linearpeptides (molecular weight generally <3 kDa), which often exhibithepatotoxic or antimicrobial properties (Okino et al., Tetrahedron Lett.34, 501-504, 1993; Krishnamurthy et al., PNAS USA 86, 770-774, 1989;Sivonen et al., Chem. Res. Toxicol. 5, 464-469, 1992; Carter et al., J.Org. Chem. 49, 236-241, 1984; and Frankmolle et al., J. Antibiot. 45,1451-1457, 1992). Sequencing studies of higher molecular weightcyanobacterial peptides and proteins have generally focused on thoseassociated with primary metabolic processes or ones that can serve asphylogenetic markers (Suter et al., FEBS Lett. 217, 279-282, 1987;Rumbeli et al., FEBS Lett. 221, 1-2, 1987; Swanson et al., J. Biol.Chem. 267, 16146-16154, 1992; Michalowski et al., Nucleic Acids Res. 18,2186, 1990; Sherman et al., in The Cyanobacteria, Fay et al., eds.,Elsevier: N.Y., 1987, pp. 1-33; and Rogers, in The Cyanobacteria, Fay etal., eds., Elsevier: N.Y., 1987, pp. 35-67). In general, proteins withantiviral properties had not been associated with cyanobacterialsources.

[0035] The cyanobacterial extract leading to the present invention wasamong many thousands of different extracts initially selected randomlyand tested blindly in the anti-HIV screen described above. A number ofthese extracts had been determined preliminarily to show anti-HIVactivity in the NCI screen (Patterson et al., J. Phycol. 29, 125-130,1993). From this group, an aqueous extract from Nostoc ellipsosporum,which had been prepared as described (Patterson, 1993, supra) and whichshowed an unusually high anti-HIV potency and in vitro “therapeuticindex” in the NCI primary screen, was selected for detailedinvestigation. A specific bioassay-guided strategy was used to isolateand purify a homogenous protein highly active against HIV.

[0036] In the bioassay-guided strategy, initial selection of the extractfor fractionation, as well as the decisions concerning the overallchemical isolation method to be applied, and the nature of theindividual steps therein, is determined by interpretation of biologicaltesting data. The anti-HIV screening assay (e.g., see Boyd, 1988, supra;Weislow et al., J. Natl. Cancer Inst. 81, 577-586, 1989), which is usedto guide the isolation and purification process, measures the degree ofprotection of human T-lymphoblastoid cells from the cytopathic effectsof HIV. Fractions of the extract of interest are prepared using avariety of chemical means and are tested blindly in the primary screen.Active fractions are separated further, and the resulting subfractionsare likewise tested blindly in the screen. This process is repeated asmany times as necessary in order to obtain the active compound(s), i.e.,antiviral fraction(s) representing pure compound(s), which then can besubjected to detailed chemical analysis and structural elucidation.

[0037] Using this strategy, aqueous extracts of Nostoc ellipsosporumwere shown to contain an antiviral protein. Accordingly, the presentinvention provides an isolated and purified antiviral protein, namedcyanovirin-N, from Nostoc ellipsosporum and functional homologs thereof,called “cyanovirins.” Herein the term “cyanovirin” is used genericallyto refer to a native cyanovirin or any related, functionally equivalent(i.e., antiviral) protein, peptide or derivative thereof. By definition,in this context, a related, functionally equivalent protein, peptide orderivative thereof a) contains a sequence of at least nine amino acidsdirectly homologous with any sub-sequence of nine contiguous amino acidscontained within a native cyanovirin, and, b) is capable of specificallybinding to a virus, in particular an influenza virus or a retrovirus,more specifically a primate immunodeficiency virus, more specificallyHIV-1, HIV-2 or SIV, or to an infected host cell expressing one or moreviral antigen(s), more specifically an envelope glycoprotein, such asgp120, of the respective virus. Herein, the term “protein” refers to asequence comprising 100 or more amino acids, whereas “peptide” refers toa sequence comprising less than 100 amino acids. In addition, such afunctionally equivalent protein or derivative thereof can comprise theamino acid sequence of a native cyanovirin, particularly cyanovirin-N(see SEQ ID NO:2), in which 1-20, preferably 1-10, more preferably 1, 2,3, 4, or 5, and most preferably 1 or 2, amino acids have been removedfrom one or both ends, preferably from only one end, and most preferablyfrom the amino-terminal end, of the native cyanovirin. Alternatively, afunctionally equivalent protein or derivative thereof can comprise theamino acid sequence of a native cyanovirin, particularly cyanovirin-N(see SEQ ID NO:2), in which 1-20, preferably 1-10, more preferably 1, 2,3, 4, or 5, and most preferably 1 or 2, amino acids have been added toone or both ends, preferably from only one end, and most preferably fromthe amino-terminal end, of the native cyanovirin.

[0038] Preferably, the protein, peptide or derivative thereof comprisesan amino acid sequence that is substantially homologous to that of anantiviral protein from Nostoc ellipsosporum. By “substantiallyhomologous” is meant sufficient homology to render the protein, peptideor derivative thereof antiviral, with antiviral activity characteristicof an antiviral protein isolated from Nostoc ellipsosporum. At leastabout 50% homology, preferably at least about 75% homology, and mostpreferably at least about 90% homology should exist. A cyanovirinconjugate comprises a cyanovirin coupled to one or more selectedeffector molecule(s), such as a toxin or immunological reagent.“Immunological reagent” will be used to refer to an antibody, animmunoglobulin, and an immunological recognition element. Animmunological recognition element is an element, such as a peptide,e.g., the FLAG sequence of the recombinant cyanovirin-FLAG fusionprotein, which facilitates, through immunological recognition, isolationand/or purification and/or analysis of the protein or peptide to whichit is attached. A cyanovirin fusion protein is a type of cyanovirinconjugate, wherein a cyanovirin is coupled to one or more otherprotein(s) having any desired properties or effector functions, such ascytotoxic or immunological properties, or other desired properties, suchas to facilitate isolation, purification or analysis of the fusionprotein.

[0039] Accordingly, the present invention provides an isolated andpurified protein encoded by a nucleic acid molecule comprising asequence of SEQ ID NO:1, a nucleic acid molecule comprising a sequenceof SEQ ID NO:3, a nucleic acid molecule encoding an amino acid sequenceof SEQ ID NO:2, or a nucleic acid molecule encoding an amino acidsequence of SEQ ID NO:4. Preferably, the aforementioned nucleic acidmolecules encode at least nine contiguous amino acids of the amino acidsequence of SEQ ID NO:2, which desirably have antiviral activity. If theat least nine contiguous amino acids of SEQ ID NO: 2 comprise aminoacids 30-32, desirably amino acids 30-32 have been renderedglycosylation resistant, yet maintain antiviral activity. Preferably,amino acids 30-32 of SEQ ID NO: 2 have been rendered glycosylationresistant by deletion or substitution of amino acid 30. Preferably,amino acid 30 has been substituted with an amino acid selected from thegroup consisting of alanine, glutamine and valine. Optionally, aminoacid 51 can be deleted or substituted, for example, with glycine.Substitution or deletion of the proline at position 51 is expected toenhance the dimerization resistance; dimerization of a cyanovirin isknown to occur under certain conditions (Yang et al., J. Molec. Biol.,288, 403-412, 1999; Bewley et al., JACS, 122, 6009-6016 (2000)), andfolding stability and resistance to physiochemical degradation aredesirable additional attributes of the cyanovirins. Such traits aredesirable for large-scale isolation and purification and mass productionof cyanovirins in prokaryotic and eukaryotic host-cells/organisms.

[0040] The present invention also provides a method of obtaining acyanovirin from Nostoc ellipsosporum. Such a method comprises (a)identifying an extract of Nostoc ellipsosporum containing antiviralactivity, (b) optionally removing high molecular weight biopolymers fromthe extract, (c) antiviral bioassay-guided fractionating the extract toobtain a crude extract of cyanovirin, and (d) purifying the crudeextract by reverse-phase HPLC to obtain cyanovirin (see, also, Example1). More specifically, the method involves the use of ethanol to removehigh molecular weight biopolymers from the extract and the use of ananti-HIV bioassay to guide fractionation of the extract.

[0041] Cyanovirin-N (a protein of exactly SEQ ID NO:2), which wasisolated and purified using the aforementioned method, was subjected toconventional procedures typically used to determine the amino acidsequence of a given pure protein. Thus, the cyanovirin was initiallysequenced by N-terminal Edman degradation of intact protein and numerousoverlapping peptide fragments generated by endoproteinase digestion.Amino acid analysis was in agreement with the deduced sequence. ESI massspectrometry of reduced, HPLC-purified cyanovirin-N showed a molecularion consistent with the calculated value. These studies indicated thatcyanovirin-N from Nostoc ellipsosporum was comprised of a uniquesequence of 101 amino acids having little or no significant homology topreviously described proteins or transcription products of knownnucleotide sequences. No more than eight contiguous amino acids fromcyanovirin were found in any amino acid sequences from known proteins,nor were there any known proteins from any source containing greaterthan 13% sequence homology with cyanovirin-N. Given the chemicallydeduced amino acid sequence of cyanovirin-N, a corresponding recombinantcyanovirin-N (r-cyanovirin-N) was created and used to establishdefinitively that the deduced amino acid sequence was, indeed, activeagainst virus, such as HIV (Boyd et al., 1995, supra; also, see Examples2-5).

[0042] Accordingly, the present invention provides isolated and purifiednucleic acid molecules and synthetic nucleic acid molecules, whichcomprise a coding sequence for a cyanovirin, such as an isolated andpurified nucleic acid molecule comprising a sequence of SEQ ID NO:1, anisolated and purified nucleic acid molecule comprising a sequence of SEQID NO:3, an isolated and purified nucleic acid molecule encoding anamino acid sequence of SEQ ID NO:2, an isolated and purified nucleicacid molecule encoding an amino acid sequence of SEQ ID NO:4, and anucleic acid molecule that is substantially homologous to any one ormore of the aforementioned nucleic acid molecules. By “substantiallyhomologous” is meant sufficient homology to render the protein, peptideor derivative thereof antiviral, with antiviral activity characteristicof an antiviral protein isolated from Nostoc ellipsosporum. At leastabout 50% homology, preferably at least about 75% homology, and mostpreferably at least about 90% homology should exist.

[0043] The present inventive nucleic acid molecule preferably comprisesa nucleic acid sequence encoding at least nine (preferably at leasttwenty, more preferably at least thirty, and most preferably at leastfifty) contiguous amino acids of the amino acid sequence of SEQ ID NO:2.The present inventive nucleic acid molecule also comprises a nucleicacid sequence encoding a protein comprising the amino acid sequence of anative cyanovirin, particularly cyanovirin-N, in which 1-20, preferably1-10, more preferably 1, 2, 3, 4, or 5, and most preferably 1 or 2,amino acids have been removed from one or both ends, preferably fromonly one end, and most preferably from the amino-terminal end, of thenative cyanovirin. Alternatively, the nucleic acid molecule can comprisea nucleic acid sequence encoding a protein comprising the amino acidsequence of a native cyanovirin, particularly cyanovirin-N (see SEQ IDNO:2), in which 1-20, preferably 1-10, more preferably 1, 2, 3, 4, or 5,and most preferably 1 or 2, amino acids have been addeded to one or bothends, preferably from only one end, and most preferably from theamino-terminal end, of the native cyanovirin. Preferably, the isolatedand purified nucleic acid molecule encodes a protein or peptidecomprising at least nine contiguous amino acids of SEQ ID NO:2, whichdesirably have antiviral activity. If the at least nine contiguous aminoacids comprise amino acids 30-32 of SEQ ID NO: 2, desirably amino acids30-32 have been rendered glycosylation resistant, yet maintain antiviralactivity. Preferably, the amino acids 30-32 of SEQ ID NO: 2 have beenrendered glycosylation resistant by deletion or substitution of aminoacid 30. Preferably, amino acid 30 has been substituted with an aminoacid selected from the group consisting of alanine, glutamine andvaline. Optionally, amino acid 51 can be deleted or substituted, forexample, with glycine. Such deletions and substitutions are within theskill in the art as indicated below.

[0044] Given the present disclosure, it will be apparent to one skilledin the art that a partial cyanovirin-N gene sequence will likely sufficeto code for a fully functional, i.e., antiviral, such as anti-influenzaor anti-HIV, cyanovirin. A minimum essential DNA coding sequence(s) fora functional cyanovirin can readily be determined by one skilled in theart, for example, by synthesis and evaluation of sub-sequencescomprising the native cyanovirin, and by site-directed mutagenesisstudies of the cyanovirin-N DNA coding sequence.

[0045] Using an appropriate DNA coding sequence, a recombinantcyanovirin can be made by genetic engineering techniques (for generalbackground see, e.g., Nicholl, in An Introduction to GeneticEngineering, Cambridge University Press: Cambridge, 1994, pp. 1-5 &127-130; Steinberg et al., in Recombinant DNA Technology Concepts andBiomedical Applications, Prentice Hall: Englewood Cliffs, N.J., 1993,pp. 81-124 & 150-162; Sofer in Introduction to Genetic Engineering,Butterworth-Heinemann, Stoneham, Mass., 1991, pp. 1-21 & 103-126; Old etal., in Principles of Gene Manipulation, Blackwell ScientificPublishers: London, 1992, pp. 1-13 & 108-221; and Emtage, in DeliverySystems for Peptide Drugs, Davis et al., eds., Plenum Press: New York,1986, pp. 23-33). For example, a Nostoc ellipsosporum gene or cDNAencoding a cyanovirin can be identified and subcloned. The gene or cDNAcan then be incorporated into an appropriate expression vector anddelivered into an appropriate protein-synthesizing organism (e.g., E.coli, S. cerevisiae, P. pastoris, or other bacterial, yeast, insect,plant or mammalian cells, where the gene, under the control of anendogenous or exogenous promoter, can be appropriately transcribed andtranslated. Alternatively, the expression vector can be administered toa plant or animal, for example, for large-scale production (see, e.g.,Fischer et al., Transzenic Res., 9(4-5), 279-299, 2000; Fischer et al.,J. Biol. Rezul. Homeost. Agents, 14, 83-92, 2000; deWilde et al., PlantMolec. Biol., 43, 347-359, 2000; Houdebine, Transgenic Research, 9,305-320, 2000; Brink et al., Theriogenolory, 53, 139-148, 2000; Pollocket al., J. Immunol. Methods, 231, 147-157, 1999; Conrad et al., PlantMolec. Biol., 38, 101-109, 1998; Staub et al., Nature Biotech., 18,333-338, 2000; McCormick et al., PNAS USA, 96, 703-708, 1999; Zeitlin etal., Nature Biotech., 16, 1361-1364 (1998); Tacker et al., Microbes andInfection, 1, 777-783, 1999; and Tacket et al., Nature Med., 4(5),607-609, 1998). Such expression vectors (including, but not limited to,phage, cosmid, viral, and plasmid vectors) are known to those skilled inthe art, as are reagents and techniques appropriate for gene transfer(e.g., transfection, electroporation, transduction, micro-injection,transformation, etc.). Subsequently, the recombinantly produced proteincan be isolated and purified using standard techniques known in the art(e.g., chromatography, centrifugation, differential solubility,isoelectric focusing, etc.), and assayed for antiviral activity. If acyanovirin is to be recombinantly produced in isolated eukaryotic cellsor in a eukaryotic organism, such as a plant (see above references andalso Methods in Biotechnology, Recombinant Proteins from Plants,Production and Isolation of Clinically Useful Compounds, Cunningham andPorter, editors, Humana Press: Totowa, N.J., 1998), desirably theN-linked glycosylation site at position 30 (Asn30-Thr31-Ser32) isrendered glycosylation-resistant, such as in accordance with the methodsdescribed herein. Optionally, amino acid 51 is deleted or substituted,for example, with glycine.

[0046] Alternatively, a native cyanovirin can be obtained from Nostocellipsosporum by non-recombinant methods (e.g., see Example 1 andabove), and sequenced by conventional techniques. The sequence can thenbe used to synthesize the corresponding DNA, which can be subcloned intoan appropriate expression vector and delivered into a protein-producingcell for en mass recombinant production of the desired protein.

[0047] In this regard, the present invention also provides a vectorcomprising a DNA sequence, e.g., a Nostoc ellipsosporum gene sequencefor cyanovirin, a cDNA encoding a cyanovirin, or a synthetic DNAsequence encoding cyanovirin, a host cell comprising the vector, and amethod of using such a host cell to produce a cyanovirin.

[0048] The DNA, whether isolated and purified or synthetic, or cDNAencoding a cyanovirin can encode for either the entire cyanovirin or aportion thereof. Where the DNA or cDNA does not comprise the entirecoding sequence of the native cyanovirin, the DNA or cDNA can besubcloned as part of a gene fusion. In a transcriptional gene fusion,the DNA or cDNA will contain its own control sequence directingappropriate production of protein (e.g., ribosome binding site,translation initiation codon, etc.), and the transcriptional controlsequences (e.g., promoter elements and/or enhancers) will be provided bythe vector. In a translational gene fusion, transcriptional controlsequences as well as at least some of the translational controlsequences (i.e., the translational initiation codon) will be provided bythe vector. In the case of a translational gene fusion, a chimericprotein will be produced.

[0049] Genes also can be constructed for specific fusion proteinscontaining a functional cyanovirin component plus a fusion componentconferring additional desired attribute(s) to the composite protein. Forexample, a fusion sequence for a toxin or immunological reagent, asdefined above, can be added to facilitate purification and analysis ofthe functional protein (e.g., such as the FLAG-cyanovirin-N fusionprotein detailed within Examples 2-5).

[0050] Genes can be specifically constructed to code for fusionproteins, which contain a cyanovirin coupled to an effector protein,such as a toxin or immunological reagent, for specific targeting to avirus or viral-infected cells, e.g., HIV and/or HIV-infected cells orinfluenza and/or influenza-infected cells. In these instances, thecyanovirin moiety serves not only as a neutralizing agent but also as atargeting agent to direct the effector activities of these moleculesselectively against a given virus, such as HIV or influenza. Thus, forexample, a therapeutic agent can be obtained by combining theHIV-targeting function or influenza-targeting function of a functionalcyanovirin with a toxin aimed at neutralizing infectious virus and/or bydestroying cells producing infectious virus, such as HIV or influenza.Similarly, a therapeutic agent can be obtained, which combines theviral-targeting function of a cyanovirin with the multivalency andeffector functions of various immunoglobulin subclasses. Example 6further illustrates the viral-targeting, specifically gp120-targeting,properties of a cyanovirin.

[0051] Similar rationales underlie extensive developmental therapeuticefforts exploiting the HIV gp120-targeting properties of sCD4. Forexample, sCD4-toxin conjugates have been prepared in which sCD4 iscoupled to a Pseudomonas exotoxin component (Chaudhary et al., in TheHuman Retrovirus, Gallo et al., eds., Academic Press: San Diego, 1991,pp. 379-387; and Chaudhary et al., Nature 335, 369-372, 1988), or to adiphtheria toxin component (Aullo et al., EMBO J. 11, 575-583, 1992) orto a ricin A-chain component (Till et al., Science 242, 1166-1167,1988). Likewise, sCD4-immunoglobulin conjugates have been prepared inattempts to decrease the rate of in vivo clearance of functional sCD4activity, to enhance placental transfer, and to effect a targetedrecruitment of immunological mechanisms of pathogen elimination, such asphagocytic engulfment and killing by antibody-dependent cell-mediatedcytotoxicity, to kill and/or remove HIV-infected cells and virus (Caponet al., Nature 337, 525-531, 1989; Traunecker et al., Nature 339, 68-70,1989; and Langner et al., 1993, supra). While such CD4-immunoglobulinconjugates (sometimes called “immunoadhesins”) have, indeed, shownadvantageous pharmacokinetic and distributional attributes in vivo, andanti-HIV effects in vitro, clinical results have been discouraging(Schooley et al., 1990, supra; Husson et al., 1992, supra; and Langneret al., 1993, supra). This is not surprising since clinical isolates ofHIV, as opposed to laboratory strains, are highly resistant to bindingand neutralization by sCD4 (Orloff et al., 1995, supra; and Moore etal., 1992, supra). Therefore, the extraordinarily broad targetingproperties of a functional cyanovirin to viruses, e.g., primateretroviruses, in general, and clinical and laboratory strains, inparticular (Boyd et al., 1995, supra; and Gustafson et al., 1995,supra), can be especially advantageous for combining with toxins,immunoglobulins and other selected effector proteins.

[0052] Viral-targeted conjugates can be prepared either by geneticengineering techniques (see, for example, Chaudhary et al., 1988, supra)or by chemical coupling of the targeting component with an effectorcomponent. The most feasible or appropriate technique to be used toconstruct a given cyanovirin conjugate or fusion protein will beselected based upon consideration of the characteristics of theparticular effector molecule selected for coupling to a cyanovirin. Forexample, with a selected non-proteinaceous effector molecule, chemicalcoupling, rather than genetic engineering techniques, may be the onlyfeasible option for creating the desired cyanovirin conjugate.

[0053] Accordingly, the present invention also provides nucleic acidmolecules encoding cyanovirin fusion proteins. In particular, thepresent invention provides a nucleic acid molecule comprising SEQ IDNO:3 and substantially homologous sequences thereof Also provided is avector comprising a nucleic acid sequence encoding a cyanovirin fusionprotein and a method of obtaining a cyanovirin fusion protein byexpression of the vector encoding a cyanovirin fusion protein in aprotein-synthesizing organism as described above. Accordingly,cyanovirin fusion proteins are also provided.

[0054] In view of the above, the present invention further provides anisolated and purified nucleic acid molecule, which comprises acyanovirin coding sequence, such as one of the aforementioned nucleicacids, namely a nucleic acid molecule encoding an amino acid sequence ofSEQ ID NO:2, a nucleic acid molecule encoding an amino acid sequence ofSEQ ID NO:4, a nucleic acid molecule comprising a sequence of SEQ IDNO:1, or a nucleic acid molecule comprising a sequence of SEQ ID NO:3,coupled to a second nucleic acid encoding an effector protein. The firstnucleic acid preferably comprises a nucleic acid sequence encoding atleast nine contiguous amino acids of the amino acid sequence of SEQ IDNO:2, which encodes a functional cyanovirin, and the second nucleic acidpreferably encodes an effector protein, such as a toxin or immunologicalreagent as described above.

[0055] Accordingly, the present invention also further provides anisolated and purified fusion protein encoded by a nucleic acid moleculecomprising a sequence of SEQ ID NO:1, a nucleic acid molecule comprisinga sequence of SEQ ID NO:3, a nucleic acid molecule encoding an aminoacid sequence of SEQ ID NO:2, or a nucleic acid molecule encoding anamino acid sequence of SEQ ID NO:4, any one of which is coupled to asecond nucleic acid encoding an effector protein. Preferably, theaforementioned nucleic acid molecules encode at least nine contiguousamino acids of the amino acid sequence of SEQ ID NO:2, which desirablyhave antiviral activity, coupled to an effector molecule, such as atoxin or immunological reagent as described above. Preferably, theeffector molecule targets a virus, more preferably HIV or influenza,and, most preferably glycoprotein gp120 of HIV. If the at least ninecontiguous amino acids of SEQ ID NO: 2 comprise amino acids 30-32,desirably amino acids 30-32 have been rendered glycosylation resistant,yet maintain antiviral activity. Preferably, amino acids 30-32 of SEQ IDNO: 2 have been rendered glycosylation resistant by deletion orsubstitution of amino acid 30. Preferably, amino acid 30 has beensubstituted with an amino acid selected from the group consisting ofalanine, glutamine and valine. Optionally, amino acid 51 can be deletedor substituted with, for example, glycine.

[0056] The coupling can be effected at the DNA level or by chemicalcoupling as described above. For example, a cyanovirin-effector proteinconjugate of the present invention can be obtained by (a) selecting adesired effector protein or peptide; (b) synthesizing a composite DNAcoding sequence comprising a first DNA coding sequence comprising one ofthe aforementioned nucleic acid sequences, which codes for a functionalcyanovirin, coupled to a second DNA coding sequence for an effectorprotein or peptide, e.g., a toxin or immunological reagent; (c)expressing said composite DNA coding sequence in an appropriateprotein-synthesizing organism; and (d) purifying the desired fusionprotein or peptide to substantially pure form. Alternatively, acyanovirin-effector molecule conjugate of the present invention can beobtained by (a) selecting a desired effector molecule and a cyanovirinor cyanovirin fusion protein; (b) chemically coupling the cyanovirin orcyanovirin fusion protein to the effector molecule; and (c) purifyingthe desired cyanovirin-effector molecule conjugate to substantially pureform.

[0057] Conjugates comprising a functional cyanovirin (e.g., an antiviralprotein or antiviral peptide comprising at least nine contiguous aminoacids of SEQ ID NO:2, such as SEQ ID NO: 2, wherein the at least ninecontiguous amino acids bind to a virus, in particular an infectiousvirus, such as influenza virus or HIV, in which case the cyanovirinbinds to gp120) coupled to an anti-cyanovirin antibody or at least oneeffector component, which can be the same or different, such as a toxin,an immunological reagent, or other functional reagent, can be designedeven more specifically to exploit the unique viral targeting, e.g.,gp120-targeting properties, of cyanovirins.

[0058] Other functional reagents that can be used as effector componentsin the present inventive conjugates can include, for example,polyethylene glycol, dextran, albumin and the like, whose intendedeffector functions may include one or more of the following: to improvestability of the conjugate; to increase the half-life of the conjugate;to increase resistance of the conjugate to proteolysis; to decrease theimmunogenicity of the conjugate; to provide a means to attach orimmobilize a functional cyanovirin onto a solid support matrix (e.g.,see, for example, Harris, in Poly(Ethylene Glycol) Chemistry:Biotechnical and Biomedical Applications, Harris, ed., Plenum Press: NewYork (1992), pp. 1-14). Conjugates furthermore can comprise a functionalcyanovirin coupled to more than one effector molecule, each of which,optionally, can have different effector functions (e.g., such as a toxinmolecule (or an immunological reagent) and a polyethylene glycol (ordextran or albumin) molecule). Diverse applications and uses offunctional proteins and peptides, such as in the present instance afunctional cyanovirin, attached to or immobilized on a solid supportmatrix, are exemplified more specifically for poly(ethylene glycol)conjugated proteins or peptides in a review by Holmberg et al. (InPoly(Ethylene Glycol) Chemistry: Biotechnical and BiomedicalApplications Harris, ed., Plenum Press: New York, 1992, pp. 303-324).Preferred examples of solid support matrices include magnetic beads, aflow-through matrix, and a matrix comprising a contraceptive device,such as a condom, a diaphragm, a cervical cap, a vaginal ring or asponge.

[0059] Example 6 reveals novel gp120-directed effects of cyanovirins.Additional insights have been reported by Esser et al. (J. Virol., 73,4360-4371, 1999). They described a series of studies that furtherconfirmed that cyanovirins block gp120-mediated binding and fusion ofintact HIV-1 virions to host cells. They also provided additionalconfirmation that cyanovirins inhibit envelope glycoprotein-mediatedinfectivity of other viruses, including feline immunodeficiency virus(FIV).

[0060] The range of antiviral activity (Boyd et al., 1995, supra) ofcyanovirin against diverse CD4⁺-tropic immunodeficiency virus strains invarious target cells is remarkable; all tested strains of HIV-1, HIV-2and SIV were similarly sensitive to cyanovirin; clinical isolates andlaboratory strains showed essentially equivalent sensitivity.Cocultivation of chronically infected and uninfected CEM-SS cells withcyanovirin did not inhibit viral replication, but did cause aconcentration-dependent inhibition of cell-to-cell fusion and virustransmission; similar results from binding and fusion inhibition assaysemploying HeLa-CD4-LTR-β-galactosidase cells were consistent withcyanovirin inhibition of virus-cell and/or cell-cell binding.

[0061] The anti-viral, e.g., anti-HIV, activity of the cyanovirins andconjugates thereof of the present invention can be further demonstratedin a series of interrelated in vitro antiviral assays (Gulakowski etal., J. Virol. Methods 33, 87-100, 1991), which accurately predict forantiviral activity in humans. These assays measure the ability ofcompounds to prevent the replication of HIV and/or the cytopathiceffects of HIV on human target cells. These measurements directlycorrelate with the pathogenesis of HIV-induced disease in vivo. Theresults of the analysis of the antiviral activity of cyanovirins orconjugates, as set forth in Examples 5 and 13 and as illustrated inFIGS. 8, 9 and 10, are believed to predict accurately the antiviralactivity of these products in vivo in humans and, therefore, establishthe utility of the present invention. Furthermore, since the presentinvention also provides methods of ex vivo use of cyanovirins andconjugates (e.g., see results set forth in Examples 5 and 13, and inFIGS. 6 and 7), the utility of cyanovirins and conjugates thereof iseven more certain.

[0062] The cyanovirins and conjugates thereof of the present inventioncan be shown to inhibit a virus, specifically a retrovirus, morespecifically an immunodeficiency virus, such as the humanimmunodeficiency virus, i.e., HIV-1 or HIV-2. The cyanovirins andconjugates of the present invention can be used to inhibit otherretroviruses as well as other viruses (see, e.g., Principles ofVirology: Molecular Biology Pathogenesis, and Control, Flint et al.,eds., ASM Press: Washington, D.C., 2000, particularly Chapter 19).Examples of viruses that may be treated in accordance with the presentinvention include, but are not limited to, Type C and Type Dretroviruses, HTLV-1, HTLV-2, HIV, FIV, FLV, SIV, MLV, BLV, BIV, equineinfectious virus, anemia virus, avian sarcoma viruses, such as Roussarcoma virus (RSV), hepatitis type A, B, non-A and non-B viruses,arboviruses, varicella viruses, human herpes virus (e.g., HHV-6),measles, mumps and rubella viruses. Cyanovirins and conjugate thereofalso can be used to inhibit influenza viral infection (see, e.g., FieldsVirology, third edition, Fields et al., eds., Lippincott-RavenPublishers: Philadelphia, Pa., 1996, particularly Chapter 45)prophylactically and therapeutically in accordance with the methods setforth herein.

[0063] Accordingly, the present invention provides a method ofinhibiting prophylactically or therapeutically a viral infection, inparticular an influenza viral infection, of a host. The method comprisesadministering to the host an effective amount of an antiviral protein,antiviral peptide, antiviral protein conjugate or antiviral peptideconjugate comprising at least nine contiguous amino acids of SEQ IDNO:2, wherein the at least nine contiguous amino acids arenonglycosylated and have antiviral activity, whereupon the viralinfection is inhibited. The antiviral protein or peptide can be derivedfrom a cyanovirin obtained from Nostoc ellipsosporum or recombinantlyproduced in accordance with the methods described above. Nonglycosylatedantiviral proteins and peptides can be produced in prokaryoticcells/organisms. Amino acid 51 in such nonglycosylated antiviralproteins and antiviral peptides can be deleted or substituted, forexample, with glycine. Nonglycosylated antiviral proteins and peptidesalso can be produced in eukaryotic cells/organisms by expressing aportion of a cyanovirin, such as that of SEQ ID NO: 2, that does notcontain a glycosylation site or all or a portion of a cyanovirin, suchas that of SEQ ID NO: 2, which contains a glycosylation site that hasbeen rendered glycosylation-resistant as described and exemplifiedherein. If the at least nine contiguous amino acids of SEQ ID NO: 2comprise amino acids 30-32, desirably amino acid 30 has been deleted orsubstituted. If amino acid 30 is substituted, preferably the amino acidis substituted with an amino acid selected from the group consisting ofalanine, glutamine and valine and, if the at least nine contiguous aminoacids of SEQ ID NO: 2 further comprises amino acid 51, optionally, aminoacid 51 is deleted or substituted, for example, with glycine. When theviral infection is an influenza viral infection, preferably theantiviral protein, antiviral peptide, antiviral protein conjugate orantiviral peptide conjugate is administered topically to the host,preferably to the respiratory system of the host, preferably as anaerosol or microparticulate powder.

[0064] Cyanovirins and conjugates thereof collectively comprise proteinsand peptides, and, as such, are particularly susceptible to hydrolysisof amide bonds (e.g., catalyzed by peptidases) and disruption ofessential disulfide bonds or formation of inactivating or unwanteddisulfide linkages (Carone et al., J. Lab. Clin. Med. 100, 1-14, 1982).There are various ways to alter molecular structure, if necessary, toprovide enhanced stability to the cyanovirin or conjugate thereof(Wunsch, Biopolymers 22, 493-505, 1983; and Samanen, in PolymericMaterials in Medication, Gebelein et al., eds., Plenum Press: New York,1985, pp. 227-242), which may be essential for preparation and use ofpharmaceutical compositions containing cyanovirins or conjugates thereoffor therapeutic or prophylactic applications against viruses, e.g., HIV.Possible options for useful chemical modifications of a cyanovirin orconjugate include, but are not limited to, the following (adapted fromSamanen, J. M., 1985, supra): (a) olefin substitution, (b) carbonylreduction, (c) D-amino acid substitution, (d) N-methyl substitution, (e)C-methyl substitution, (f) C-C′-methylene insertion, (g) dehydro aminoacid insertion, (h) retro-inverso modification, (i) N-terminal toC-terminal cyclization, and () thiomethylene modification. Cyanovirinsand conjugates thereof also can be modified by covalent attachment ofcarbohydrate and polyoxyethylene derivatives, which are expected toenhance stability and resistance to proteolysis (Abuchowski et al., inEnzymes as Drugs, Holcenberg et al., eds., John Wiley: New York, 1981,pp. 367-378).

[0065] Other important general considerations for design of deliverystrategy systems and compositions, and for routes of administration, forprotein and peptide drugs, such as cyanovirins and conjugates thereof(Eppstein, CRC Crit. Rev. Therapeutic Drug Carrier Systems 5, 99-139,1988; Siddiqui et al., CRC Crit. Rev. Therapeutic Drug Carrier Systems3, 195-208, 1987); Banga et al., Int. J. Pharmaceutics 48, 15-50, 1988;Sanders, Eur. J. Drug Metab. Pharmacokinetics 15, 95-102, 1990; andVerhoef, Eur. J. Drug Metab. Pharmacokinetics 15, 83-93, 1990), alsoapply. The appropriate delivery system for a given cyanovirin orconjugate thereof will depend upon its particular nature, the particularclinical application, and the site of drug action. As with any proteinor peptide drug, oral delivery of a cyanovirin or a conjugate thereofwill likely present special problems, due primarily to instability inthe gastrointestinal tract and poor absorption and bioavailability ofintact, bioactive drug therefrom. Therefore, especially in the case oforal delivery, but also possibly in conjunction with other routes ofdelivery, it will be necessary to use an absorption-enhancing agent incombination with a given cyanovirin or conjugate thereof. A wide varietyof absorption-enhancing agents have been investigated and/or applied incombination with protein and peptide drugs for oral delivery and fordelivery by other routes (Verhoef, 1990, supra; van Hoogdalem, Pharmac.Ther. 44, 407-443, 1989; Davis, J. Pharm. Pharmacol. 44(Suppl. 1),186-190, 1992). Most commonly, typical enhancers fall into the generalcategories of (a) chelators, such as EDTA, salicylates, and N-acylderivatives of collagen, (b) surfactants, such as lauryl sulfate andpolyoxyethylene-9-lauryl ether, (c) bile salts, such as glycholate andtaurocholate, and derivatives, such as taurodihydrofusidate, (d) fattyacids, such as oleic acid and capric acid, and their derivatives, suchas acylcarnitines, monoglycerides and diglycerides, (e) non-surfactants,such as unsaturated cyclic ureas, (f) saponins, (g) cyclodextrins, and(h) phospholipids.

[0066] Other approaches to enhancing oral delivery of protein andpeptide drugs, such as the cyanovirins and conjugates thereof, caninclude aforementioned chemical modifications to enhance stability togastrointestinal enzymes and/or increased lipophilicity. Alternatively,or in addition, the protein or peptide drug can be administered incombination with other drugs or substances, which directly inhibitproteases and/or other potential sources of enzymatic degradation ofproteins and peptides. Yet another alternative approach to prevent ordelay gastrointestinal absorption of protein or peptide drugs, such ascyanovirins or conjugates, is to incorporate them into a delivery systemthat is designed to protect the protein or peptide from contact with theproteolytic enzymes in the intestinal lumen and to release the intactprotein or peptide only upon reaching an area favorable for itsabsorption. A more specific example of this strategy is the use ofbiodegradable microcapsules or microspheres, both to protect vulnerabledrugs from degradation, as well as to effect a prolonged release ofactive drug (Deasy, in Microencapsulation and Related Processes,Swarbrick, ed., Marcell Dekker, Inc.: New York, 1984, pp. 1-60, 88-89,208-211). Microcapsules also can provide a useful way to effect aprolonged delivery of a protein and peptide drug, such as a cyanovirinor conjugate thereof, after injection (Maulding, J. Controlled Release6, 167-176, 1987).

[0067] Given the aforementioned potential complexities of successfuloral delivery of a protein or peptide drug, it is fortunate that thereare numerous other potential routes of delivery of a protein or peptidedrug, such as a cyanovirin or conjugate thereof. These routes includeintravenous, intraarterial, intrathecal, intracisternal, buccal, rectal,nasal, pulmonary, transdermal, vaginal, ocular, and the like (Eppstein,1988, supra; Siddiqui et al., 1987, supra; Banga et al., 1988, supra;Sanders, 1990, supra; Verhoef, 1990, supra; Barry, in Delivery Systemsfor Peptide Drugs, Davis et al., eds., Plenum Press: New York, 1986, pp.265-275; and Patton et al., Adv. Drug Delivery Rev. 8, 179-196, 1992).With any of these routes, or, indeed, with any other route ofadministration or application, a protein or peptide drug, such as acyanovirin or conjugate thereof, may initiate an immunogenic reaction.In such situations it may be necessary to modify the molecule in orderto mask immunogenic groups. It also can be possible to protect againstundesired immune responses by judicious choice of method of formulationand/or administration. For example, site-specific delivery can beemployed, as well as masking of recognition sites from the immune systemby use or attachment of a so-called tolerogen, such as polyethyleneglycol, dextran, albumin, and the like (Abuchowski et al., 1981, supra;Abuchowski et al., J. Biol. Chem. 252, 3578-3581, 1977; Lisi et al., J.Appl. Biochem. 4, 19-33, 1982; and Wileman et al., J. Pharm. Pharmacol.38, 264-271, 1986). Such modifications also can have advantageouseffects on stability and half-life both in vivo and ex vivo.

[0068] Procedures for covalent attachment of molecules, such aspolyethylene glycol, dextran, albumin and the like, to proteins, such ascyanovirins or conjugates thereof, are well-known to those skilled inthe art, and are extensively documented in the literature (e.g., seeDavis et al., In Peptide and Protein Drug Delivery, Lee, ed., MarcelDekker: New York, 1991, pp. 831-864).

[0069] Other strategies to avoid untoward immune reactions can alsoinclude the induction of tolerance by administration initially of onlylow doses. In any event, it will be apparent from the present disclosureto one skilled in the art that for any particular desired medicalapplication or use of a cyanovirin or conjugate thereof, the skilledartisan can select from any of a wide variety of possible compositions,routes of administration, or sites of application, what is advantageous.

[0070] Accordingly, the antiviral cyanovirins and conjugates thereof ofthe present invention can be formulated into various compositions foruse, for example, either in therapeutic treatment methods for infectedindividuals, or in prophylactic methods against viral, e.g., HIV andinfluenza virus, infection of uninfected individuals.

[0071] The present invention also provides a composition, such as apharmaceutical composition, which comprises an isolated and purifiedcyanovirin, a cyanovirin conjugate, a matrix-anchored cyanovirin or amatrix-anchored cyanovirin conjugate, such as an antiviral effectiveamount thereof. The composition can further comprise a carrier, such asa pharmaceutically acceptable carrier. The composition can furthercomprise at least one additional antiviral compound other than acyanovirin or conjugate thereof, such as in an antiviral effectiveamount. Suitable antiviral compounds include AZT, ddI, ddC, gancyclovir,fluorinated dideoxynucleosides, nevirapine, R82913, Ro 31-8959,BI-RJ-70, acyclovir, α-interferon, recombinant sCD4, michellamines,calanolides, nonoxynol-9, gossypol and derivatives thereof, andgramicidin. If the composition is to be used to induce an immuneresponse, it comprises an immune response-inducing amount of acyanovirin or conjugate thereof and can further comprise animmunoadjuvant, such as polyphosphazene polyelectrolyte. The cyanovirinused in the composition, e.g., pharmaceutical composition, can beisolated and purified from nature or genetically engineered. Similarly,the cyanovirin conjugate can be genetically engineered or chemicallycoupled.

[0072] The present inventive compositions can be administered to a host,such as a human, so as to inhibit viral infection in a prophylactic ortherapeutic method. The compositions of the present invention areparticularly useful in inhibiting the growth or replication of a virus,such as influenza virus or a retrovirus, in particular animmunodeficiency virus, such as HIV, specifically HIV-1 and HIV-2. Thecompositions are useful in the therapeutic or prophylactic treatment ofanimals, such as humans, who are infected with a virus or who are atrisk for viral infection, respectively. The compositions also can beused to treat objects or materials, such as medical equipment, supplies,or fluids, including biological fluids, such as blood, blood productsand vaccine formulations, cells, tissues and organs, to remove orinactivate virus in an effort to prevent or treat viral infection of ananimal, such as a human. Such compositions also are useful to preventsexual transmission of viral infections, e.g., HIV, which is the primaryway in which the world's AIDS cases are contracted (Merson, 1993,supra).

[0073] Potential virucides used or being considered for use againstsexual transmission of HIV are very limited; present agents in thiscategory include, for example, nonoxynol-9 (Bird, AIDS 5, 791-796,1991), gossypol and derivatives (Polsky et al., Contraception 39,579-587, 1989; Lin, Antimicrob. Agents Chemother. 33, 2149-2151, 1989;and Royer, Pharmacol. Res. 24, 407-412, 1991), and gramicidin(Bourinbair, Life Sci./Pharmacol. Lett. 54, PL5-9, 1994; and Bourinbairet al., Contraception 49, 131-137, 1994). The method of prevention ofsexual transmission of viral infection, e.g., HIV infection, inaccordance with the present invention comprises vaginal, rectal, oral,penile or other topical treatment with an antiviral effective amount ofa cyanovirin and/or cyanovirin conjugate, alone or in combination withanother antiviral compound as described above.

[0074] In a novel approach to anti-HIV prophylaxis pursued underauspices of the U.S. National Institute of Allergy and InfectiousDiseases (NIAID) (e.g., as conveyed by Painter, USA Today, Feb. 13,1996), vaginal suppository instillation of live cultures of lactobacilliwas being evaluated in a 900-woman study. This study was basedespecially upon observations of anti-HIV effects of certainH₂O₂-producing lactobacilli in vitro (e.g., see published abstract byHilier, from NIAID-sponsored Conference on “Advances in AIDS VaccineDevelopment”, Bethesda, Md., Feb. 11-15, 1996). Lactobacilli readilypopulate the vagina, and indeed are a predominant bacterial populationin most healthy women (Redondo-Lopez et al., Rev. Infect. Dis. 12,856-872, 1990; Reid et al., Clin. Microbiol. Rev. 3, 335-344, 1990;Bruce and Reid, Can. J. Microbiol. 34, 339-343, 1988;reu et al., J.Infect. Dis. 171, 1237-1243, 1995; Hilier et al., Clin. Infect. Dis.16(Suppl 4), S273-S281; Agnew et al., Sex. Transm. Dis. 22, 269-273,1995). Lactobacilli are also prominent, nonpathogenic inhabitants ofother body cavities such as the mouth, nasopharynx, upper and lowergastrointestinal tracts, and rectum.

[0075] It is well-established that lactobacilli can be readilytransformed using available genetic engineering techniques toincorporate a desired foreign DNA coding sequence, and that suchlactobacilli can be made to express a corresponding desired foreignprotein (see, e.g., Hols et al., Appl. and Environ. Microbiol. 60,1401-1413, 1994). Therefore, within the context of the presentdisclosure, it will be appreciated by one skilled in the art that viablehost cells containing a DNA sequence or vector of the present invention,and expressing a protein of the present invention, can be used directlyas the delivery vehicle for a cyanovirin or conjugate thereof to thedesired site(s) in vivo. Preferred host cells for such delivery ofcyanovirins or conjugates thereof directly to desired site(s), such as,for example, to a selected body cavity, can comprise bacteria. Morespecifically, such host cells can comprise suitably engineered strain(s)of lactobacilli, enterococci, or other common bacteria, such as E. coli,normal strains of which are known to commonly populate body cavities.More specifically yet, such host cells can comprise one or more selectednonpathogenic strains of lactobacilli, such as those described by Andreuet al. (1995, supra), especially those having high adherence propertiesto epithelial cells, such as, for example, adherence to vaginalepithelial cells, and suitably transformed using the DNA sequences ofthe present invention.

[0076] As reviewed by McGroarty (FEMS Immunol. Med. Microbiol. 6,251-264, 1993) the “probiotic” or direct therapeutic application of livebacteria, particularly bacteria that occur normally in nature, moreparticularly lactobacilli, for treatment or prophylaxis againstpathogenic bacterial or yeast infections of the urogenital tract, inparticular the female urogenital tract, is a well-established concept.Recently, the use of a conventional probiotic strategy, in particularthe use of live lactobacilli, to inhibit sexual transmission of HIV hasbeen suggested, based specifically upon the normal, endogenousproduction of virucidal levels of H₂O₂ and/or lactic acid and/or otherpotentially virucidal substances by certain normal strains oflactobacilli (e.g., Hilier, 1996, supra). However, the present inventiveuse of non-mammalian cells, particularly bacteria, more particularlylactobacilli, specifically engineered with a foreign gene, morespecifically a cyanovirin gene, to express an antiviral substance, morespecifically a protein, and even more specifically a cyanovirin, isheretofore unprecedented as a method of treatment of an animal,specifically a human, to prevent infection by a virus, specifically aretrovirus, more specifically HIV-1 or HIV-2.

[0077] Elmer et al. (JAMA 275, 870-876, 1996) have recently speculatedthat “genetic engineering offers the possibility of using microbes todeliver specific actions or products to the colon or other mucosalsurfaces . . . other fertile areas for future study include defining themechanisms of action of various biotherapeutic agents with thepossibility of applying genetic engineering to enhance activities.”Elmer et al. (1996, supra) further point out that the terms “probiotic”and “biotherapeutic agent” have been used in the literature to describemicroorganisms that have antagonistic activity toward pathogens in vivo;those authors more specifically prefer the term “biotherapeutic agent”to denote “microorganisms having specific therapeutic properties.

[0078] In view of the present disclosure, one skilled in the art willappreciate that the present invention teaches an entirely novel type of“probiotic” or “biotherapeutic” treatment using specifically engineeredstrains of microorganisms provided herein which do not occur in nature.Nonetheless, available teachings concerning selection of optimalmicrobial strains, in particular bacterial strains, for conventionalprobiotic or biotherapeutic applications can be employed in the contextof the present invention. For example, selection of optimallactobacillus strains for genetic engineering, transformation, directexpression of cyanovirins or conjugates thereof, and direct probiotic orbiotherapeutic applications, to treat or prevent HIV infection, can bebased upon the same or similar criteria, such as those described byElmer et al. (1996, supra , typically used to select normal, endogenousor “nonengineered” bacterial strains for conventional probiotic orbiotherapeutic therapy. Furthermore, the recommendations andcharacteristics taught by McGroarty, particularly for selection ofoptimal lactobacillus strains for conventional probiotic use againstfemale urogenital infections, are pertinent to the present invention: “. . . lactobacilli chosen for incorporation into probiotic preparationsshould be easy and, if possible, inexpensive to cultivate . . . strainsshould be stable, retain viability following freeze-drying and, ofcourse, be non-pathogenic to the host . . . it is essential thatlactobacilli chosen for use in probiotic preparations should adhere wellto the vaginal epithelium . . . ideally, artificially implantedlactobacilli should adhere to the vaginal epithelium, integrate with theindigenous microorganisms present, and proliferate” (McGroarty, 1993supra). While McGroarty's teachings specifically address selections of“normal” lactobacillus strains for probiotic uses against pathogenicbacterial or yeast infections of the female urogenital tract, similarconsiderations will apply to the selection of optimal bacterial strainsfor genetic engineering and “probiotic” or “biotherapeutic” applicationagainst viral infections as particularly encompassed by the presentinvention.

[0079] Accordingly, the method of the present invention for theprevention of sexual transmission of viral infection, e.g., HIVinfection, comprises vaginal, rectal, oral, penile, or other topical,insertional, or instillational treatment with an antiviral effectiveamount of a cyanovirin, a cyanovirin conjugate, a matrix-anchoredcyanovirin or conjugate thereof, and/or viable host cells transformed toexpress a cyanovirin or conjugate thereof, alone or in combination withone or more other antiviral compound (e.g., as described above).

[0080] Compositions for use in the prophylactic or therapeutic treatmentmethods of the present invention comprise one or more cyanovirin(s) orconjugate(s) thereof, either one of which can be matrix-anchored, anddesirably a carrier therefor, such as a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are well-known to thosewho are skilled in the art, as are suitable methods of administration.The choice of carrier will be determined in part by the particularcyanovirin or conjugate thereof, as well as by the particular methodused to administer the composition.

[0081] One skilled in the art will appreciate that various routes ofadministering a drug are available, and, although more than one routecan be used to administer a particular drug, a particular route canprovide a more immediate and more effective reaction than another route.Furthermore, one skilled in the art will appreciate that the particularpharmaceutical carrier employed will depend, in part, upon theparticular cyanovirin or conjugate thereof employed, and the chosenroute of administration. Accordingly, there is a wide variety ofsuitable formulations of the composition of the present invention.

[0082] Formulations suitable for oral administration can consist ofliquid solutions, such as an effective amount of the compound dissolvedin diluents, such as water, saline, or fruit juice; capsules, sachets ortablets, each containing a predetermined amount of the activeingredient, as solid, granules or freeze-dried cells; solutions orsuspensions in an aqueous liquid; and oil-in-water emulsions orwater-in-oil emulsions. Tablet forms can include one or more of lactose,mannitol, corn starch, potato starch, microcrystalline cellulose,acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc,magnesium stearate, stearic acid, and other excipients, colorants,diluents, buffering agents, moistening agents, preservatives, flavoringagents, and pharmacologically compatible carriers. Suitable formulationsfor oral delivery can also be incorporated into synthetic and naturalpolymeric microspheres, or other means to protect the agents of thepresent invention from degradation within the gastrointestinal tract(see, for example, Wallace et al., Science 260, 912-915, 1993).

[0083] The cyanovirins or conjugates thereof, alone or in combinationwith other antiviral compounds, can be made into aerosol formulations ormicroparticulate powder formulations to be administered via inhalation.These aerosol formulations can be placed into pressurized acceptablepropellants, such as dichlorodifluoromethane, propane, nitrogen and thelike.

[0084] The cyanovirins or conjugates thereof, alone or in combinationswith other antiviral compounds or absorption modulators, can be madeinto suitable formulations for transdermal application and absorption(Wallace et al., 1993, supra). Transdermal electroporation oriontophoresis also can be used to promote and/or control the systemicdelivery of the compounds and/or compositions of the present inventionthrough the skin (e.g., see Theiss et al., Meth. Find. Exp. Clin.Pharmacol. 13, 353-359, 1991).

[0085] Formulations suitable for topical administration include lozengescomprising the active ingredient in a flavor, usually sucrose and acaciaor tragacanth; pastilles comprising the active ingredient in an inertbase, such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the active ingredient in a suitable liquidcarrier; as well as creams, emulsions, gels and the like containing, inaddition to the active ingredient, such as, for example, freeze-driedlactobacilli or live lactobacillus cultures genetically engineered todirectly produce a cyanovirin or conjugate thereof of the presentinvention, such carriers as are known in the art. Topical administrationis preferred for the prophylactic and therapeutic treatment of influenzaviral infection, such as through the use of an inhaler, for example.

[0086] Formulations for rectal administration can be presented as asuppository with a suitable base comprising, for example, cocoa butteror a salicylate. Formulations suitable for vaginal administration can bepresented as pessaries, tampons, creams, gels, pastes, foams, or sprayformulas containing, in addition to the active ingredient, such as, forexample, freeze-dried lactobacilli or live lactobacillus culturesgenetically engineered to directly produce a cyanovirin or conjugatethereof of the present invention, such carriers as are known in the artto be appropriate. Similarly, the active ingredient can be combined witha lubricant as a coating on a condom. Indeed, preferably, the activeingredient is applied to any contraceptive device, including, but notlimited to, a condom, a diaphragm, a cervical cap, a vaginal ring and asponge.

[0087] Formulations suitable for parenteral administration includeaqueous and non-aqueous, isotonic sterile injection solutions, which cancontain anti-oxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

[0088] Formulations comprising a cyanovirin or cyanovirin conjugatesuitable for virucidal (e.g., HIV) sterilization of inanimate objects,such as medical supplies or equipment, laboratory equipment andsupplies, instruments, devices, and the like, can, for example, beselected or adapted as appropriate.! by one skilled in the art, from anyof the aforementioned compositions or formulations. Preferably, thecyanovirin is produced by recombinant DNA technology. The cyanovirinconjugate can be produced by recombinant DNA technology or by chemicalcoupling of a cyanovirin with an effector molecule as described above.Similarly, formulations suitable for ex vivo sterilization or removal ofvirus, such as infectious virus, from a sample, such as blood, bloodproducts, sperm, or other bodily products, such as a fluid, cells, atissue or an organ, or any other solution, suspension, emulsion, vaccineformulation (such as in the removal of infectious virus), or any othermaterial which can be administered to a patient in a medical procedure,can be selected or adapted as appropriate by one skilled in the art,from any of the aforementioned compositions or formulations. However,suitable formulations for ex vivo sterilization or removal of virus froma sample or on an inanimate object are by no means limited to any of theaforementioned formulations or compositions. For example, suchformulations or compositions can comprise a functional cyanovirin, suchas that which is encoded by SEQ ID NO: 2, or antiviral fragment thereof,such as a fragment comprising at least nine contiguous amino acids ofSEQ ID NO:2, wherein the at least nine contiguous amino acids bind to avirus, or a conjugate of either of the foregoing, attached to a solidsupport matrix, to facilitate contacting or binding infectious virus ina sample or removing infectious virus from a sample as described above,e.g., a bodily product such as a fluid, cells, a tissue or an organ froman organism, in particular a mammal, such as a human, including, forexample, blood, a component of blood, or sperm. Preferably, theantiviral protein comprises SEQ ID NO: 2. Also preferably, the at leastnine contiguous amino acids bind gp120 of HIV, in particular infectiousHIV. As a more specific example, such a formulation or composition cancomprise a functional cyanovirin, or conjugate thereof, attached to(e.g., coupled to or immobilized on) a solid support matrix comprisingmagnetic beads, to facilitate contacting, binding and removal ofinfectious virus, and to enable magnet-assisted removal of the virusfrom a sample as described above, e.g., a bodily product such as afluid, cells, a tissue or an organ, e.g., blood, a component of blood,or sperm. Alternatively, and also preferably, the solid support matrixcomprises a contraceptive device, such as a condom, a diaphragm, acervical cap, a vaginal ring or a sponge.

[0089] As an even more specific illustration, such a composition (e.g.,for ex vivo) can comprise a functional (e.g., gp120-binding,HIV-inactivating) cyanovirin, or conjugate thereof, attached to a solidsupport matrix, such as magnetic beads or a flow-through matrix, bymeans of an anti-cyanovirin antibody or at least one effector component,which can be the same or different, such as polyethylene glycol, albuminor dextran. The conjugate can further comprise at least one effectorcomponent, which can be the same or different, selected from the groupconsisting of an immunological reagent and a toxin. A flow-throughmatrix would comprise, for instance, a configuration similar to anaffinity column. The cyanovirin can be covalently coupled to a solidsupport matrix via an anti-cyanovirin antibody, described below. Methodsof attaching an antibody to a solid support matrix are well-known in theart (see, for example, Harlow and Lane. Antibodies: A Laboratory Manual,Cold Springs Harbor Laboratory: Cold Spring Harbor, N.Y., 1988).Alternatively, the solid support matrix, such as magnetic beads, can becoated with streptavidin, in which case the cyanovirin or fragmentthereof (which comprises at least nine contiguous amino acids of SEQ IDNO: 2), or a conjugate of either one, is biotinylated. The at least ninecontiguous amino acids of SEQ ID NO: 2 desirably have antiviral activityand preferably bind gp120 of HIV, which preferably is infectious.Preferably, the antiviral protein comprises SEQ ID NO: 2. Such acomposition can be prepared, for example, by biotinylating thecyanovirin, or conjugate thereof, and then contacting the biotinylatedprotein or peptide with a (commercially available) solid support matrix,such as magnetic beads, coated with streptavidin. The use ofbiotinylation as a means to attach a desired biologically active proteinor peptide to a streptavidin-coated support matrix, such as magneticbeads, is well-known in the art. Example 7 specifically describes aprocedure applicable to biotinylation of cyanovirin-N and attachment ofthe biotinylated protein to streptavidin-coated magnetic beads. Example7 also illustrates another important principle that is critical to thepresent invention: for any given formulation or composition comprising afunctional cyanovirin, or conjugate thereof, attached to or immobilizedon a solid support matrix, it is essential that the formulation orcomposition retain the desired (i.e., in this instance, the virusbinding (e.g., gp120-binding) and virus-inactivating (e.g., HIV))properties of the functional cyanovirin, or conjugate thereof, itself.

[0090] One skilled in the art will appreciate that a suitable orappropriate formulation can be selected, adapted or developed based uponthe particular application at hand.

[0091] For ex vivo uses, such as virucidal treatments of inanimateobjects or materials, blood or blood products, or tissues, the amount ofcyanovirin, or conjugate or composition thereof, to be employed shouldbe sufficient that any virus or virus-producing cells present will berendered noninfectious or will be destroyed. For example, for HIV, thiswould require that the virus and/or the virus-producing cells be exposedto concentrations of cyanovirin-N in the range of 0.1-1000 nM. Similarconsiderations apply to in vivo applications. Therefore, the designationof “antiviral effective amount” is used generally to describe the amountof a particular cyanovirin, conjugate or composition thereof requiredfor antiviral efficacy in any given application.

[0092] In view of the above, the present invention also provides amethod of inhibiting prophylactically or therapeutically a viralinfection of a host in which an antiviral effective amount of anabove-described conjugate is administered to the host. Uponadministration of the antiviral effective amount of the conjugate, theviral infection is inhibited.

[0093] The present invention additionally provides a method ofprophylactically or therapeutically inhibiting a viral infection of ahost in which an antiviral effective amount of a composition comprisingan isolated and purified antiviral protein, antiviral peptide, antiviralprotein conjugate or antiviral peptide conjugate comprising at leastnine contiguous amino acids of SEQ ID NO: 2 having antiviral activityand attached to a solid support matrix is administered to the host. Uponadministration of the antiviral effective amount of the composition, theviral infection is inhibited. Preferably, the solid support matrix is acontraceptive device, such as a condom, diaphragm, cervical cap, vaginalring or sponge. In an alternative embodiment, a solid support matrix canbe surgically implanted and later removed.

[0094] For in vivo uses, the dose of a cyanovirin, or conjugate orcomposition thereof, administered to an animal, particularly a human, inthe context of the present invention should be sufficient to effect aprophylactic or therapeutic response in the individual over a reasonabletime frame. The dose used to achieve a desired antiviral concentrationin vivo (e.g., 0.1-1000 nM) will be determined by the potency of theparticular cyanovirin or conjugate employed, the severity of the diseasestate of infected individuals, as well as, in the case of systemicadministration, the body weight and age of the infected individual. Thesize of the dose also will be determined by the existence of any adverseside effects that may accompany the particular cyanovirin, or conjugateor composition thereof, employed. It is always desirable, wheneverpossible, to keep adverse side effects to a minimum.

[0095] The present invention also provides a method of removing virus,such as infectious virus, from a sample. The method comprises contactingthe sample with a composition comprising an isolated and purifiedantiviral protein, antiviral peptide, antiviral protein conjugate, orantiviral peptide conjugate, comprising at least nine contiguous aminoacids of SEQ ID NO:2. The at least nine contiguous amino acids desirablyhave antiviral activity and bind to the virus and the antiviral proteinor antiviral peptide (or conjugate of either of the foregoing) isattached to a solid support matrix, such as a magnetic bead. “Attached”is used herein to refer to attachment to (or coupling to) andimmobilization in or on a solid support matrix. While any means ofattachment can be used, preferably, attachment is by covalent bonds. Themethod further comprises separating the sample and the composition byany suitable means, whereupon the virus, such as infectious virus, isremoved from the sample. Preferably, the antiviral protein comprises SEQID NO:2. In one embodiment, the antiviral protein or antiviral peptideis conjugated with an anti-cyanovirin antibody or at least one effectorcomponent, which can be the same or different, selected frompolyethylene glycol, dextran and albumin, in which case the antiviralprotein or antiviral peptide is desirably attached to the solid supportmatrix through at least one effector component. The antiviral protein orantiviral peptide can be further conjugated with at least one effectorcomponent, which can be the same or different, selected from the groupconsisting of an immunological reagent and a toxin. In anotherembodiment, the solid support matrix is coated with streptavidin and theantiviral protein or antiviral peptide is biotinylated. Through biotin,the biotinylated antiviral protein/peptide is attached to thestreptavidin-coated solid support matrix. Other types of means, as areknown in the art, can be used to attach a functional cyanovirin (i.e.,an antiviral protein or peptide or conjugate of either of the foregoingas described above) to a solid support matrix, such as a magnetic bead,in which case contact with a magnet is used to separate the sample andthe composition. Similarly, other types of solid support matrices can beused, such as a matrix comprising a porous surface or membrane, over orthrough which a sample is flowed or percolated, thereby selectivelyentrapping or removing infectious virus from the sample. The choice ofsolid support matrix, means of attachment of the functional cyanovirinto the solid support matrix, and means of separating the sample and thematrix-anchored cyanovirin will depend, in part, on the sample (e.g.,fluid vs. tissue) and the virus to be removed. It is expected that theuse of a selected coupling molecule can confer certain desiredproperties to a matrix, comprising a functional cyanovirin coupledtherewith, that may have particularly advantageous properties in a givensituation. Preferably, the sample is blood, a component of blood, sperm,cells, tissue or an organ. Also, preferably the sample is a vaccineformulation, in which case the virus that is removed is infectious, suchas HIV, although HIV, in particular infectious HIV, can be removed fromother samples in accordance with this method. Such methods also haveutility in real time ex vivo removal of virus or virus infected cellsfrom a bodily fluid, such as blood, e.g., in the treatment of viralinfection, or in the removal of virus from blood or a component ofblood, e.g., for transfusion, in the inhibition or prevention of viralinfection. Such methods also have potential utility in dialysis, such askidney dialysis, and in removing virus from sperm obtained from a donorfor in vitro and in vivo fertilization. The methods also haveapplicability in the context of tissue and organ transplantations.

[0096] For instance, the skilled practitioner might select apoly(ethylene glycol) molecule for attaching a functional cyanovirin toa solid support matrix, thereby to provide a matrix-anchored cyanovirin,wherein the cyanovirin is attached to the matrix by a longer “tether”than would be feasible or possible for other attachment methods, such asbiotinylation/streptavidin coupling. A cyanovirin coupled by apoly(ethylene glycol) “tether” to a solid support matrix (such asmagnetic beads, porous surface or membrane, and the like) can permitoptimal exposure of a binding surface, epitope, hydrophobic orhydrophobic focus, and/or the like, on a functional cyanovirin in amanner that, in a given situation and/or for a particular virus,facilitates the binding and/or inactivation of the virus. A preferredsolid support matrix is a magnetic bead such that separation of thesample and the composition is effected by a magnet. In a preferredembodiment of the method, the at least nine contiguous amino acids bindgp120 of HIV and HIV is removed from the sample.

[0097] In summary, a cyanovirin attached to a solid support matrix, suchas a magnetic bead, can be used to remove virus, in particularinfectious virus, including immunodeficiency virus, such as HIV, e.g.,HIV-1 or HIV-2, from a sample, such as a sample comprising bothinfectious and noninfectious virus. In contrast to previously disclosedmethods of inactivating or destroying virus in a sample, the presentinventive method utilizes cyanovirins, which bind irreversibly to virus,such as infectious virus, in particular infectious immunodeficiencyvirus, e.g., HIV, specifically HIV-1 or HIV-2. The infectious viralparticle is permanently fixed to the antiviral agent and unable toinfect host cells. The present inventive method enables separation ofinfectious from non-infectious virus and, therefore, is advantageousover other methods currently available in the art. In this regard, ithas been unexpectedly observed that a cyanovirin recognizes aconformation or a portion of gp120 characteristic of infectious HIV.Therefore, only infectious virus is bound by the cyanovirin, whilenon-infectious virus remains in the sample. One of ordinary skill in theart will appreciate the advantages of using the present inventivematrix-anchored cyanovirins to produce pools of non-infectious virus.The present inventive method also can be used to remove gp120-presentingcells, e.g., infected cells that have gp120 on their surfaces, from asample.

[0098] The present invention, therefore, further provides a compositioncomprising naturally-occurring, non-infectious virus, such as acomposition produced as described above. The composition can furthercomprise a carrier, such as a biologically or pharmaceuticallyacceptable carrier, and an immuno-adjuvant. Preferably, thenoninfectious virus is an immunodeficiency virus, such as HIV, e.g.,HIV-1 or HIV-2. Alternatively, and also preferably, the noninfectiousvirus is FIV. A composition comprising only naturally-occurring,non-infectious virus has many applications in research and theprophylactic treatment of a viral infection. In terms of prophylactictreatment of a viral infection, the skilled artisan will appreciate theneed to eliminate completely all infectious virus from the composition.If desired, further treatment of the composition comprisingnon-infectious particles with virus-inactivating chemicals, such asimines or psoralens, and/or pressure or heat inactivation, will furtherthe non-infectious nature of the composition. For example, an immuneresponse-inducing amount of the present inventive composition can beadministered to an animal at risk for a viral infection in order toinduce an immune response. The skilled artisan will appreciate that sucha composition is a significant improvement over previously disclosedcompositions in that the virus is non-infectious andnaturally-occurring. Thus, there is no risk of inadvertent infection,greater doses can be administered in comparison to compositionscomprising infectious viral particles, and the subsequent immuneresponse will assuredly be directed to antigens present onnaturally-occurring virus. The composition comprisingnaturally-occurring, non-infectious virus can be administered in anymanner appropriate to induce an immune response. Preferably, the virusis administered, for example, intramuscularly, mucosally, intravenously,subcutaneously, or topically. Preferably, the composition comprisesnaturally-occurring, non-infectious human immunodeficiency viruscomprising gp120.

[0099] The composition comprising naturally-occuring, non-infectiousvirus can be combined with various carriers, adjuvants, diluents orother anti-viral therapeutics, if desired. Appropriate carriers include,for example, ovalbumin, albumin, globulins, hemocyanins, and the like.Adjuvants or immuno-adjuvants are incorporated in most cases tostimulate further the immune system. Any physiologically appropriateadjuvant can be used. Suitable adjuvants for inclusion in the presentinventive composition include, for example, aluminum hydroxide,beryllium sulfate, silica, kaolin, carbon, bacterial endotoxin, saponin,and the like.

[0100] Thus, the present invention also provides a method of inducing animmune response to a virus in an animal. The method comprisesadministering to the animal an immune response-inducing amount of acomposition comprising naturally-occurring, non-infectious virus asdescribed above.

[0101] The appropriate dose of a composition comprisingnaturally-occuring, non-infectious virus required to induce an immuneresponse to the virus in an animal is dependent on numerous factors,such as size of the animal and immune competency. The amount ofcomposition administered should be sufficient to induce a humoral and/orcellular immune response. The amount of non-infectious virus in aparticular composition can be determined using routine methods in theart, such as the Coulter HIV p24 antigen assay (Coulter Corp., Hialeah,Fla.). Any suitable dose of a composition comprising non-infectiousvirus is appropriate so long as an immune response is induced, desirablywithout the appearance of harmful side effects to the host. In thisregard, compositions comprising from about 10¹ to about 10⁵ particles,preferably from about 10² to about 10⁴ particles, most preferably about10³ particles, are suitable for inducing an immune response.

[0102] One of ordinary skill can determine the effectiveness of thecomposition to induce an immune response using routine methods known inthe art. Cell-mediated response can be determined by employing, forexample, a virus antigen-stimulated T-cell proliferation assay. Thepresence of a humoral immune response can be determined, for instance,with the Enzyme Linked Immunosorbent Assay (ELISA). The skilled artisanwill appreciate that there are numerous other suitable assays forevaluating induction of an immune response. To the extent that a dose isinadequate to induce an appropriate immune response, “booster”administrations can subsequently be administered in order to prompt amore effective immune response.

[0103] In terms of administration of the present inventive antiviralagents or conjugates thereof, the dosage can be in unit dosage form,such as a tablet or capsule. The term “unit dosage form” as used hereinrefers to physically discrete units suitable as unitary dosages forhuman and animal subjects, each unit containing a predetermined quantityof a cyanovirin or conjugate thereof, alone or in combination with otherantiviral agents, calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptablediluent, carrier, or vehicle.

[0104] The specifications for the unit dosage forms of the presentinvention depend on the particular cyanovirin, or conjugate orcomposition thereof, employed and the effect to be achieved, as well asthe pharmacodynamics associated with each cyanovirin, or conjugate orcomposition thereof, in the host. The dose administered should be an“antiviral effective amount” or an amount necessary to achieve an“effective level” in the individual patient.

[0105] Since the “effective level” is used as the preferred endpoint fordosing, the actual dose and schedule can vary, depending uponinterindividual differences in pharmacokinetics, drug distribution, andmetabolism. The “effective level” can be defined, for example, as theblood or tissue level (e.g., 0.1-1000 nM) desired in the patient thatcorresponds to a concentration of one or more cyanovirin or conjugatethereof, which inhibits a virus, such as HIV, in an assay known topredict for clinical antiviral activity of chemical compounds andbiological agents. The “effective level” for agents of the presentinvention also can vary when the cyanovirin, or conjugate or compositionthereof, is used in combination with AZT or other known antiviralcompounds or combinations thereof.

[0106] One skilled in the art can easily determine the appropriate dose,schedule, and method of administration for the exact formulation of thecomposition being used, in order to achieve the desired “effectiveconcentration” in the individual patient. One skilled in the art alsocan readily determine and use an appropriate indicator of the “effectiveconcentration” of the compounds of the present invention by a direct(e.g., analytical chemical analysis) or indirect (e.g., with surrogateindicators such as p24 or RT) analysis of appropriate patient samples(e.g., blood and/or tissues).

[0107] In the treatment of some virally infected individuals, it can bedesirable to utilize a “mega-dosing” regimen, wherein a large dose ofthe cyanovirin or conjugate thereof is administered, time is allowed forthe drug to act, and then a suitable reagent is administered to theindividual to inactivate the drug.

[0108] The pharmaceutical composition can contain other pharmaceuticals,in conjunction with the cyanovirin or conjugate thereof, when used totherapeutically treat a viral infection, such as that which results inAIDS. Representative examples of these additional pharmaceuticalsinclude antiviral compounds, virucides, immunomodulators,immunostimulants, antibiotics and absorption enhancers. Exemplaryantiviral compounds include AZT, ddI, ddC, gancylclovir, fluorinateddideoxynucleosides, nonnucleoside analog compounds, such as nevirapine(Shih et al., PNAS 88, 9878-9882, 1991), TIBO derivatives, such asR82913 (White et al., Antiviral Res. 16, 257-266, 1991), BI-RJ-70(Merigan, Am. J. Med. 90 (Suppl.4A), 8S-17S, 1991), michellamines (Boydet al., J. Med. Chem. 37, 1740-1745, 1994) and calanolides (Kashman etal., J. Med. Chem. 35, 2735-2743, 1992), nonoxynol-9, gossypol andderivatives, and gramicidin (Bourinbair et al., 1994, supra). Exemplaryimmunomodulators and immunostimulants include various interleukins,sCD4, cytokines, antibody preparations, blood transfusions, and celltransfusions. Exemplary antibiotics include antifungal agents,antibacterial agents, and anti-Pneumocystitis carnii agents. Exemplaryabsorption enhancers include bile salts and other surfactants, saponins,cyclodextrins, and phospholipids (Davis, 1992, supra).

[0109] Administration of a cyanovirin or conjugate thereof with otheranti-retroviral agents and particularly with known RT inhibitors, suchas ddC, AZT, ddI, ddA, or other inhibitors that act against other HIVproteins, such as anti-TAT agents, is expected to inhibit most or allreplicative stages of the viral life cycle. The dosages of ddC and AZTused in AIDS or ARC patients have been published. A virustatic range ofddC is generally between 0.05 μM to 1.0 μM. A range of about 0.005-0.25mg/kg body weight is virustatic in most patients. The preliminary doseranges for oral administration are somewhat broader, for example 0.001to 0.25 mg/kg given in one or more doses at intervals of 2, 4, 6, 8, 12,etc. hours. Currently, 0.01 mg/kg body weight ddC given every 8 hrs ispreferred. When given in combined therapy, the other antiviral compound,for example, can be given at the same time as the cyanovirin orconjugate thereof or the dosing can be staggered as desired. The twodrugs also can be combined in a composition. Doses of each can be lesswhen used in combination than when either is used alone.

[0110] It will also be appreciated by one skilled in the art that a DNAsequence of a cyanovirin or conjugate thereof of the present inventioncan be inserted ex vivo into mammalian cells previously removed from agiven animal, in particular a human, host. Such cells can be employed toexpress the corresponding cyanovirin or conjugate in vivo afterreintroduction into the host. Feasibility of such a therapeutic strategyto deliver a therapeutic amount of an agent in close proximity to thedesired target cells and pathogens, i.e., virus, more particularlyretrovirus, specifically HIV and its envelope glycoprotein gp120, hasbeen demonstrated in studies with cells engineered ex vivo to expresssCD4 (Morgan et al., 1994, supra). It is also possible that, as analternative to ex vivo insertion of the DNA sequences of the presentinvention, such sequences can be inserted into cells directly in vivo,such as by use of an appropriate viral vector. Such cells transfected invivo are expected to produce antiviral amounts of cyanovirin or aconjugate thereof directly in vivo.

[0111] Given the present disclosure, it will be additionally appreciatedthat a DNA sequence corresponding to a cyanovirin or conjugate thereofcan be inserted into suitable nonmammalian host cells, and that suchhost cells will express therapeutic or prophylactic amounts of acyanovirin or conjugate thereof directly in vivo within a desired bodycompartment of an animal, in particular a human. Example 3 illustratesthe transformation and expression of effective virucidal amounts of acyanovirin in a non-mammalian cell, more specifically a bacterial cell.Example 10 illustrates the transformation and expression of a cyanovirinin a non-mammalian cell, specifically a yeast cell. If a yeast cell isto be transformed, desirably the cyanovirin is nonglycosylated orrendered glycosylation-resistant as described above.

[0112] In a preferred embodiment of the present invention, a method offemale-controllable prophylaxis against HIV infection comprises theintravaginal administration and/or establishment of, in a female human,a persistent intravaginal population of lactobacilli that have beentransformed with a coding sequence of the present invention to produce,over a prolonged time, effective virucidal levels of a cyanovirin orconjugate thereof, directly on or within the vaginal and/or cervicaland/or uterine mucosa. It is noteworthy that both the World HealthOrganization (WHO), as well as the U.S. National Institute of Allergyand Infectious Diseases, have pointed to the need for development offemale-controlled topical microbicides, suitable for blocking thetransmission of HIV, as an urgent global priority (Lange et al., Lancet341, 1356, 1993; Fauci, NIAID News, Apr. 27, 1995). A compositioncomprising a present inventive antiviral agent and a solid-supportmatrix is particularly useful in this regard, particularly when thesolid-support matrix is a contraceptive device, such as a condom, adiaphragm, a cervical cap, a vaginal ring, or a sponge.

[0113] The present invention also provides antibodies directed to theproteins of the present invention. The availability of antibodies to anygiven protein is highly advantageous, as it provides the basis for awide variety of qualitative and quantitative analytical methods,separation and purification methods, and other useful applicationsdirected to the subject proteins. Accordingly, given the presentdisclosure and the proteins of the present invention, it will be readilyapparent to one skilled in the art that antibodies, in particularantibodies specifically binding to a protein of the present invention,can be prepared using well-established methodologies (e.g., such as themethodologies described in detail by Harlow and Lane, in Antibodies. ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,1988, pp. 1-725). Such antibodies can comprise both polyclonal andmonoclonal antibodies. Furthermore, such antibodies can be obtained andemployed either in solution-phase or coupled to a desired solid-phasematrix, such as magnetic beads or a flow through matrix. Having in handsuch antibodies as provided by the present invention, one skilled in theart will further appreciate that such antibodies, in conjunction withwell-established procedures (e.g., such as described by Harlow and Lane(1988, supra) comprise useful methods for the detection, quantification,or purification of a cyanovirin, conjugate thereof, or host celltransformed to produce a cyanovirin or conjugate thereof. Example 12further illustrates an antibody specifically binding a cyanovirin.

[0114] Matrix-anchored anti-cyanovirin antibodies also can be used in amethod to remove virus in a sample. Preferably, the antibody binds to anepitope of an antiviral protein or an antiviral peptide comprising atleast nine contiguous amino acids of SEQ ID NO: 2. Preferably, thematrix is a solid support matrix, such as a magnetic bead or aflow-through matrix. If the solid support matrix to which theanti-cyanovirin antibody is attached comprises magnetic beads, removalof the antibody-cyanovirin-virus complex can be readily accomplishedusing a magnet.

[0115] In view of the above, the present invention provides a method ofremoving virus from a sample. The method comprises (a) contacting thesample with a composition comprising an isolated and purified antiviralprotein, antiviral peptide, antiviral protein conjugate or antiviralpeptide conjugate, wherein (i) the antiviral protein or the antiviralpeptide comprises at least nine contiguous amino acids of SEQ ID NO: 2,and (ii) the at least nine contiguous amino acids bind to the virus, and(b) contacting the sample with an anti-cyanovirin antibody attached to asolid support matrix, whereupon the anti-cyanovirin antibody binds tothe antiviral peptide, antiviral protein, antiviral peptide conjugate orantiviral protein conjugate to which is bound the virus, and (c)separating the solid support matrix from the sample, whereupon the virusis removed from the sample. Preferably, the antiviral protein comprisesSEQ ID NO: 2. Desirably, the virus that is removed is infectious, suchas HIV. The sample can be blood, a component of blood, sperm, cells,tissue or an organ.

[0116] The antibody for use in the aforementioned method is an antibodythat binds to a protein or a peptide comprising at least nine contiguousamino acids of SEQ ID NO: 2, and, which protein or peptide can bind toand inactivate a virus. The antibody can be coupled to the solid supportmatrix using similar methods and with similar considerations asdescribed above for attaching a cyanovirin to a solid support matrix.For example, coupling methods and molecules employed to attach ananti-cyanovirin antibody to a solid support matrix, such as magneticbeads or a flow-through matrix, can employ biotin/streptavidin couplingor coupling through molecules, such as polyethylene glycol, albumin ordextran. For instance, essentially the same procedure as described inExample 7 for attaching a cyanovirin to a solid support matrixcomprising magnetic beads can be used to attach an anti-cyanovirinantibody to magnetic beads. Also analogously, it can be shown that,after such coupling, the matrix-anchored anti-cyanovirin antibodyretains its ability to bind to a protein or a peptide comprising atleast nine contiguous amino acids of SEQ ID NO:2, which protein orpeptide can bind to and inactivate a virus.

[0117] The present inventive cyanovirins, conjugates, host cells,antibodies, compositions and methods are further described in thecontext of the following examples. These examples serve to illustratefurther the present invention and are not intended to limit the scope ofthe invention.

EXAMPLES Example 1

[0118] This example shows details of anti-HIV bioassay-guided isolationand elucidation of pure cyanovirin from aqueous extracts of the culturedcyanobacterium, Nostoc ellipsosporum.

[0119] The method described in Weislow et al. (1989, supra) was used tomonitor and direct the isolation and purification process.Cyanobacterial culture conditions, media and classification were asdescribed previously (Patterson, J. Phycol. 27, 530-536, 1991). Briefly,the cellular mass from a unialgal strain of Nostoc ellipsosporum(culture Q68D170) was harvested by filtration, freeze-dried andextracted with MeOH-CH₂Cl₂ (1:1) followed by H₂0. Bioassay indicatedthat only the H₂0 extract contained HIV-inhibitory activity. A solutionof the aqueous extract (30 mg/ml) was treated by addition of an equalvolume of ethanol (EtOH). The resulting 1:1 H₂0-EtOH solution was keptat −20° C. for 15 hrs. Then, the solution was centrifuged to removeprecipitated materials (presumably, high molecular weight biopolymers).The resulting HIV-inhibitory supernatant was evaporated, thenfractionated by reverse-phase vacuum-liquid chromatography (Coll et al.,J. Nat. Prod. 49, 934-936, 1986; and Pelletier et al., J. Nat. Prod. 49,892-900, 1986) on wide-pore C₄ packing (300, BakerBond WP-C₄), andeluted with increasing concentrations of methanol (MeOH) in H₂0.Anti-HIV activity was concentrated in the material eluted with MeOH-H₂0(2:1). SDS-PAGE analysis of this fraction showed one main protein band,with a relative molecular mass (Mr) of approximately 10 kDa. Finalpurification was achieved by repeated reverse-phase HPLC on 1.9×15 cmμBondapak C₁₈ (Waters Associates) columns eluted with a gradient ofincreasing concentration of acetonitrile in H₂0. The mobile phasecontained 0.05% (v/v) TFA, pH=2. Eluted proteins and peptides weredetected by UV absorption at 206, 280 and 294 nm with a rapid spectraldetector (Pharmacia LKB model 2140). Individual fractions werecollected, pooled based on the UV chromatogram, and lyophilized. PooledHPLC fractions were subjected to SDS-PAGE under reducing conditions(Laemmli, Nature 227, 680-685, 1970), conventional amino acid analysis,and testing for anti-HIV activity.

[0120]FIG. 1A is a graph of OD 206 nm versus time (min), which shows theμBondapak C₁₈ HPLC chromatogram of nonreduced cyanovirin eluted with alinear CH₃CN/H₂0 gradient (buffered with 0.05% TFA) from 28-38% CH₃CN.FIG. 1C is a graph of OD 206 nm versus time (min), which shows thechromatogram of cyanovirin that was first reduced with β-mercaptoethanoland then separated under identical HPLC conditions. HPLC fractions fromthe two runs were collected as indicated. 10% aliquots of each fractionwere lyophilized, made up in 100 μl 3:1 H₂0/DMSO and assessed foranti-HIV activity in the XTT assay. FIG. 1B is a bar graph of maximumdilution for 50% protection versus HPLC fraction, which illustrates themaximum dilution of each fraction that provided 50% protection from thecytopathic effects of HIV infection for the nonreduced cyanovirin HPLCfractions. Corresponding anti-HIV results for the HPLC fractions fromreduced cyanovirin are shown in FIG. 1D, which is a bar graph of maximumdilution for 50% protection versus HPLC fraction. 20% aliquots ofselected HPLC fractions were analyzed by SDS-PAGE. In the initial HPLCseparation, using a linear gradient from 30-50% CH₃CN, the anti-HIVactivity coeluted with the principal UV-absorbing absorbing peak atapproximately 33% CH₃CN. Fractions corresponding to the active peak werepooled and split into two aliquots.

[0121] Reinjection of the first aliquot under similar HPLC conditions,but with a linear gradient from 28-38% CH₃CN, resolved the activematerial into two closely eluting peaks at 33.4 and 34.0% CH₃CN. Theanti-HIV activity profile of the fractions collected during this HPLCrun (as shown in FIG. 1B) corresponded with the two UV peaks (as shownin FIG. 1A). SDS-PAGE of fractions collected under the individual peaksshowed only a single protein band.

[0122] The second aliquot from the original HPLC separation was reducedwith β-mercaptoethanol prior to reinjection on the HPLC. Using anidentical 28-38% gradient, the reduced material gave one principal peak(as shown in FIG. 1C) that eluted later in the run with 36.8% CH₃CN.Only a trace of anti-HIV activity was detected in the HPLC fractionsfrom the reduced material (as shown in FIG. 1D).

[0123] The two closely eluting HPLC peaks of the nonreduced material(FIG. 1A) gave only one identical band on SDS-PAGE (run under reducingconditions), and reduction with β-mercaptoethanol resulted in an HPLCpeak with a longer retention time than either of the nonreduced peaks.This indicated that disulfides were present in the native protein. Aminoacid analysis of the two active peaks showed they had virtuallyidentical compositions. It is possible that the two HPLC peaks resultedfrom cis/trans isomerism about a proline residue or frommicroheterogeneity in the protein sample that was not detected in eitherthe amino acid analysis or during sequencing. The material collected asthe two HIV-inhibitory peaks was combined for further analyses and wasgiven the name cyanovirin-N.

Example 2

[0124] This example illustrates synthesis of cyanovirin genes.

[0125] The chemically deduced amino acid sequence of cyanovirin-N wasback-translated to obtain a DNA coding sequence. In order to facilitateinitial production and purification of recombinant cyanovirin-N, acommercial expression vector (pFLAG-1, from InternationalBiotechnologies, Inc., New Haven, Conn.), for which reagents wereavailable for affinity purification and detection, was selected.Appropriate restriction sites for ligation to pFLAG-1, and a stop codon,were included in the DNA sequence. FIG. 2 is an example of a DNAsequence encoding a synthetic cyanovirin gene. This DNA sequence designcouples the cyanovirin-N coding region to codons for a “FLAG”octapeptide at the N-terminal end of cyanovirin, providing forproduction of a FLAG-cyanovirin fusion protein.

[0126] A flowchart for synthesis of this DNA sequence is shown in FIG.11. The DNA sequence was synthesized as 13 overlapping, complementaryoligonucleotides and assembled to form the double-stranded codingsequence. Oligonucleotide elements of the synthetic DNA coding sequencewere synthesized using a dual-column nucleic acid synthesizer (Model392, Applied Biosystems Inc., Foster City, Calif.). Completedoligonucleotides were cleaved from the columns and deprotected byincubation overnight at 56° C. in concentrated ammonium hydroxide. Priorto treatment with T4 polynucleotide kinase, 33-66 mers weredrop-dialyzed against distilled water. The 13 oligonucleotidepreparations were individually purified by HPLC, and 10 nmole quantitiesof each were ligated with T4 DNA ligase into a 327 bp double-strandedDNA sequence. DNA was recovered and purified from the reaction buffer byphenol:chloroform extraction, ethanol precipitation, and further washingwith ethanol. Individual oligonucleotide preparations were pooled andboiled for 10 min to ensure denaturation. The temperature of the mixturewas then reduced to 70° C. for annealing of the complementary strands.After 20 min, the tube was cooled on ice and 2,000 units of T4 DNAligase were added together with additional ligase buffer. Ligation wasperformed overnight at 16° C. DNA was recovered and purified from theligation reaction mixture by phenol:chloroform extraction and ethanolprecipitation and washing.

[0127] The purified, double-stranded synthetic DNA was then used as atemplate in a polymerase chain reaction (PCR). One μl of the DNAsolution obtained after purification of the ligation reaction mixturewas used as a template. Thermal cycling was performed using aPerkin-Elmer instrument. “Vent” thermostable DNA polymerase, restrictionenzymes, T4 DNA ligase and polynucleotide -kinase were obtained from NewEngland Biolabs, Beverly, Mass. Vent polymerase was selected for thisapplication because of its claimed superiority in fidelity compared tothe usual Taq enzyme. The PCR reaction product was run on a 2% agarosegel in TBE buffer. The 327 bp construct was then cut from the gel andpurified by electroelution. Because it was found to be relativelyresistant to digestion with Hind III and Xho I restriction enzymes, itwas initially cloned using the pCR-Script system (Stratagene). Digestionof a plasmid preparation from one of these clones yielded the codingsequence, which was then ligated into the multicloning site of thepFLAG-1 vector.

[0128]E. coli were transformed with the pFLAG-construct and recombinantclones were identified by analysis of restriction digests of plasmidDNA. Sequence analysis of one of these selected clones indicated thatfour bases deviated from the intended coding sequence. This includeddeletion of three bases coding for one of four cysteine residuescontained in the protein and an alteration of the third base in thepreceding codon (indicated by the boxes in FIG. 2). In order to correctthese “mutations,” which presumably arose during the PCR amplificationof the synthetic template, a double-stranded “patch” was synthesized,which could be ligated into restriction sites flanking the mutations(these Bst XI and Esp1 sites are also indicated in FIG. 2). The patchwas applied and the repair was confirmed by DNA sequence analysis.

[0129] For preparation of a DNA sequence coding for native cyanovirin,the aforementioned FLAG-cyanovirin construct was subjected tosite-directed mutagenesis to eliminate the codons for the FLAGoctapeptide and, at the same time, to eliminate a unique Hind IIIrestriction site. This procedure is illustrated in FIG. 3, whichillustrates a site-directed mutagenesis maneuver used to eliminatecodons for a FLAG octapeptide and a Hind III restriction site from thesequence of FIG. 2. A mutagenic oligonucleotide primer was synthesized,which included portions of the codons for the Omp secretory peptide andcyanovirin, but lacking the codons for the FLAG peptide. Annealing ofthis mutagenic primer, with creation of a DNA hairpin in the templatestrand, and extension by DNA polymerase resulted in generation of newplasmid DNA lacking both the FLAG codon sequence and the Hind III site(refer to FIG. 2 for details). Digestion of plasmid DNA with Hind IIIresulted in linearization of “wild-type” strands but not “mutant”strands. Since transformation of E. coli occurs more efficiently withcircular DNA, clones could be readily selected which had the revisedcoding sequence which specified production of native cyanovirin-Ndirectly behind the Omp secretory peptide. DNA sequencing verified thepresence of the intended sequence. Site-directed mutagenesis reactionswere carried out using materials (polymerase, buffers, etc.) obtainedfrom Pharmacia Biotech, Inc., Piscataway, N.J.

Example 3

[0130] This example illustrates expression of synthetic cyanoviringenes.

[0131]E. coli (strain DH5α) were transformed (by electroporation) withthe pFLAG-1 vector containing the coding sequence for theFLAG-cyanovirin-N fusion protein (see FIG. 2 for details of the DNAsequence). Selected clones were seeded into small-scale shake flaskscontaining (LB) growth medium with 100 μg/ml ampicillin and expanded byincubation at 37° C. Larger-scale Erlenmeyer flasks (0.5-3.0 liters)were then seeded and allowed to grow to a density of 0.5-0.7 OD₆₀₀units. Expression of the FLAG-cyanovirin-N fusion protein was theninduced by adding IPTG to a final concentration of 1.7 mM and continuingincubation at 30° C. for 3-6 hrs. For harvesting of periplasmicproteins, bacteria were pelleted, washed, and then osmotically shockedby treatment with sucrose, followed by resuspension in distilled water.Periplasmic proteins were obtained by sedimenting the bacteria and thenfiltering the aqueous supernatant through Whatman paper. Crudeperiplasmic extracts showed both anti-HIV activity and presence of aFLAG-cyanovirin-N fusion protein by Western or spot-blotting.

[0132] The construct for native cyanovirin-N described in Example 2 wasused to transform bacteria in the same manner as described above for theFLAG-cyanovirin-N fusion protein. Cloning, expansion, induction withIPTG, and harvesting were performed similarly. Crude periplasmicextracts showed strong anti-HIV activity on bioassay. A flowchart of theexpression of synthetic cyanovirin genes is shown in FIG. 12.

Example 4

[0133] This example illustrates purification of recombinant cyanovirinproteins.

[0134] Using an affinity column based on an anti-FLAG monoclonalantibody (International Biotechnologies, Inc., New Haven, Conn.),FLAG-cyanovirin-N fusion protein could be purified as follows.

[0135] The respective periplasmic extract, prepared as described inExample 3, was loaded onto 2-20 ml gravity columns containing affinitymatrix and washed extensively with PBS containing CA⁺⁺ to removecontaminating proteins. Since the binding of the FLAG peptide to theantibody is Ca⁺⁺-dependent, fusion protein could be eluted by passage ofEDTA through the column. Column fractions and wash volumes weremonitored by spot-blot analysis using the same anti-FLAG antibody.Fractions containing fusion protein were then pooled, dialyzedextensively against distilled water, and lyophilized.

[0136] For purification of recombinant native cyanovirin-N, thecorresponding periplasmic extract from Example 3 was subjected tostep-gradient C₄ reverse-phase, vacuum-liquid chromatography to givethree fractions: (1) eluted with 100% H₂0, (2) eluted with MeOH-H₂0(2:1), and (3) eluted with 100% MeOH. The anti-HIV activity wasconcentrated in fraction (2). Purification of the recombinantcyanovirin-N was performed by HPLC on a 1.9×15 cm μBondapak (WatersAssociates) C₁₈ column eluted with a gradient of increasingconcentration of CH₃CN in H₂0 (0.05% TFA, v/v in the mobile phase). Achromatogram of the final HPLC purification on a 1×10 cm (CohensiveTechnologies, Inc.) C₄ column monitored at 280 nm is shown in FIG. 4,which is typical HPLC chromatogram during the purification of arecombinant native cyanovirin. Gradient elution, 5 ml/min, from 100% H₂0to H₂0-CH₃CN (7:3) was carried out over 23 min with 0.05% TFA (v/v) inthe mobile phase.

Example 5

[0137] This example shows anti-HIV activities of natural and recombinantcyanovirin-N and FLAG-cyanovirin-N.

[0138] Pure proteins were initially evaluated for antiviral activityusing an XTT-tetrazolium anti-HIV assay described previously (Boyd, inAIDS, Etiology, Diagnosis, Treatment and Prevention, 1988, supra;Gustafson et al., J. Med. Chem. 35, 1978-1986, 1992; Weislow, 1989,supra; and Gulakowski, 1991, supra). The CEM-SS human lymphocytic targetcell line used in all assays was maintained in RPMI 1650 medium (Gibco,Grand Island, N.Y.), without phenol red, and was supplemented with 5%fetal bovine serum, 2 mM L-glutamine, and 50 μg/ml gentamicin (completemedium).

[0139] Exponentially growing cells were pelleted and resuspended at aconcentration of 2.0×10⁵ cells/ml in complete medium. The Haitianvariant of HIV, HTLV-III_(RF) (3.54×10⁶ SFU/ml), was used throughout.Frozen virus stock solutions were thawed immediately before use andresuspended in complete medium to yield 1.2×12⁵ SFU/ml. The appropriateamounts of the pure proteins for anti-HIV evaluations were dissolved inH₂0-DMSO (3:1), then diluted in complete medium to the desired initialconcentration. All serial drug dilutions, reagent additions, andplate-to-plate transfers were carried out with an automated Biomek 1000Workstation (Beckman Instruments, Palo Alto, Calif.).

[0140]FIGS. 5A-5C are graphs of % control versus concentration (nm),which illustrate antiviral activities of native cyanovirin from Nostocellipsosporum (A), recombinant native (B), and recombinant FLAG-fusion(C) cyanovirins. The graphs show the effects of a range ofconcentrations of the respective cyanovirins upon CEM-SS cells infectedwith HIV-1 (), as determined after 6 days in culture. Data pointsrepresent the percent of the respective uninfected, nondrug-treatedcontrol values. All three cyanovirins showed potent anti-HIV activity,with an EC₅₀ in the low nanomolar range and no significant evidence ofdirect cytotoxicity to the host cells at the highest testedconcentrations (up to 1.2 μM).

[0141] As an example of a further demonstration of the anti-HIV activityof pure cyanovirin-N, a battery of interrelated anti-HIV assays wasperformed in individual wells of 96-well microtiter plates, usingmethods described in detail elsewhere (Gulakowski, 1991, supra).Briefly, the procedure was as follows. Cyanovirin solutions wereserially diluted in complete medium and added to 96-well test plates.Uninfected CEM-SS cells were plated at a density of 1×10⁴ cells in 50 μlof complete medium. Diluted HIV-1 was then added to appropriate wells ina volume of 50 μl to yield a multiplicity of infection of 0.6.Appropriate cell, virus, and drug controls were incorporated in eachexperiment. The final volume in each microtiter well was 200 μl.Quadruplicate wells were used for virus-infected cells. Plates wereincubated at 37° C. in an atmosphere containing 5% CO₂ for 4, 5, or 6days.

[0142] Subsequently, aliquots of cell-free supernatant were removed fromeach well using the Biomek, and analyzed for reverse transcriptaseactivity, p24 antigen production, and synthesis of infectious virions asdescribed (Gulakowski, 1991, supra). Cellular growth or viability thenwas estimated on the remaining contents of each well using the XTT(Weislow et al., 1989, supra), BCECF (Rink et al., J. Cell Biol. 95,189-196, 1982), and DAPI (McCaffrey et al., In Vitro Cell Develop. Biol.24, 247-252, 1988) assays as described (Gulakowski et al., 1991, supra).To facilitate graphical displays and comparisons of data, the individualexperimental assay results (of at least quadruplicate determinations ofeach) were averaged, and the mean values were used to calculatepercentages in reference to the appropriate controls. Standard errors ofthe mean values used in these calculations typically averaged less than10% of the respective mean values.

[0143]FIGS. 6A-6D are graphs of % control versus concentration (nm),which illustrate anti-HIV activity of a cyanovirin in a multiparameterassay format. Graphs 6A, 6B, and 6C show the effects of a range ofconcentrations of cyanovirin upon uninfected CEM-SS cells (◯), and uponCEM-SS cells infected with HIV-1 (), as determined after 6 days inculture. Graph 6A depicts the relative numbers of viable CEM-SS cells,as assessed by the BCECF assay. Graph 6B depicts the relative DNAcontents of the respective cultures. Graph 6C depicts the relativenumbers of viable CEM-SS cells, as assessed by the XTT assay. Graph 6Dshows the effects of a range of concentrations of cyanovirin uponindices of infectious virus or viral replication as determined after 4days in culture. These indices include viral reverse transcriptase (▴),viral core protein p24 (♦), and syncytium-forming units (▪). In graphs6A, 6B, and 6C, the data are represented as the percent of theuninfected, nondrug-treated control values. In graph 6D the data arerepresented as the percent of the infected, nondrug-treated controlvalues.

[0144] As illustrated in FIG. 6, cyanovirin-N was capable of completeinhibition of the cytopathic effects of HIV-1 upon CEM-SS humanlymphoblastoid target cells in vitro; direct cytotoxicity of the proteinupon the target cells was not observed at the highest testedconcentrations. Cyanovirin-N also strikingly inhibited the production ofRT, p24, and SFU in HIV-1-infected CEM-SS cells within these sameinhibitory effective concentrations, indicating that the protein haltedviral replication.

[0145] The anti-HIV activity of the cyanovirins is extremely resilientto harsh environmental challenges. For example, unbuffered cyanovirin-Nsolutions withstood repeated freeze-thaw cycles or dissolution inorganic solvents (up to 100% DMSO, MeOH, or CH₃CN) with no loss ofactivity. Cyanovirin-N tolerated detergent (0.1% SDS), high salt (6 Mguanidine HCl) and heat treatment (boiling, 10 min in H₂0) with nosignificant loss of HIV inhibitory activity. Reduction of the disulfideswith β-mercaptoethanol, followed immediately by C₁₈ HPLC purification,drastically reduced the cytoprotective activity of cyanovirin-N.However, solutions of reduced cyanovirin-N regained anti-HIV inhibitoryactivity during prolonged storage. When cyanovirin-N was reduced(□mercaptoethanol, 6 M guanidine HCl, pH 8.0) but not put through C₁₈HPLC, and, instead, simply desalted, reconstituted and assayed, itretained virtually full activity.

Example 6

[0146] This example illustrates that the HIV viral envelope gp120 is aprincipal molecular target of cyanovirin-N.

[0147] Initial experiments, employing the XTT-tetrazolium assay (Weislowet al., 1989, supra), revealed that host cells preincubated withcyanovirin (10 nM, 1 hr), then centrifuged free of cyanovirin-N,retained normal susceptibility to HIV infection; in contrast, theinfectivity of concentrated virus similarly pretreated, then diluted toyield non-inhibitory concentrations of cyanovirin-N, was essentiallyabolished. This indicated that cyanovirin-N was acting directly upon thevirus itself, i.e., acting as a direct “virucidal” agent to preventviral infectivity even before it could enter the host cells. This wasfurther confirmed in time-of-addition experiments, likewise employingthe XTT-tetrazolium assay (Weislow, 1989, supra), which showed that, toafford maximum antiviral activity, cyanovirin-N had to be added to cellsbefore or as soon as possible after addition of virus as shown in FIG.7, which is a graph of % uninfected control versus time of addition(hrs), which shows results of time-of-addition studies of a cyanovirin,showing anti-HIV activity in CEM-SS cells infected with HIV-1_(RF).Introduction of cyanovirin () or ddC (▪) (10 nM and 5 μMconcentrations, respectively) was delayed by various times after initialincubation, followed by 6 days incubation, then assay of cellularviability (linegraphs) and RT (open bars, inset). Points representaverages (±S.D.) of at least triplicate determinations. In markedcontrast to the reverse transcriptase inhibitor ddC, delay of additionof cyanovirin-N by only 3 hrs resulted in little or no antiviralactivity (FIG. 7). The aforementioned results suggested thatcyanovirin-N inhibited HIV-infectivity by interruption of the initialinteraction of the virus with the cell; this would, therefore, likelyinvolve a direct interaction of cyanovirin-N with the viral gp120. Thiswas confirmed by ultrafiltration experiments and dot-blot assays.

[0148] Ultrafiltration experiments were performed to determine ifsoluble gp120 and cyanovirin-N could bind directly, as assessed byinhibition of passage of cyanovirin-N through a 50 kDa-cutoffultrafilter. Solutions of cyanovirin (30 μg) in PBS were treated withvarious concentrations of gp120 for 1 hr at 37° C., then filteredthrough a 50 kDa-cutoff centrifugal ultrafilter (Amicon). After washing3 times with PBS, filtrates were desalted with 3 kDa ultrafilters;retentates were lyophilized, reconstituted in 100 μl H₂0 and assayed foranti-HIV activity.

[0149]FIG. 8 is a graph of OD (450 nm) versus cyanovirin concentration(μg/ml), which illustrates cyanovirin/gp120 interactions defining gp120as a principal molecular target of cyanovirin. Free cyanovirin-N wasreadily eluted, as evidenced by complete recovery of cyanovirin-Nbioactivity in the filtrate. In contrast, filtrates from cyanovirin-Nsolutions treated with gp120 revealed a concentration-dependent loss offiltrate bioactivity; moreover, the 50 kDa filter retentates were allinactive, indicating that cyanovirin-N and soluble gp120 interacteddirectly to form a complex incapable of binding to gp120 of intactvirus.

[0150] There was further evidence of a direct interaction ofcyanovirin-N and gp120 in a PVDF membrane dot-blot assay. A PVDFmembrane was spotted with 5 μg CD4 (CD), 10 μg aprotinin (AP), 10 μgbovine globulin (BG), and decreasing amounts of cyanovirin; 6 μg [1], 3μg [2], 1.5 μg [3], 0.75 μg [4], 0.38 μg [5], 0.19 μg [6], 0.09 μg [7],and 0.05 μg [8], then washed with PBST and visualized per manufacturer'srecommendations. A dot blot of binding of cyanovirin and a gp120-HRPconjugate (Invitrogen) showed that cyanovirin-N specifically bound ahorseradish peroxidase conjugate of gp120 (gp120-HRP) in aconcentration-dependent manner.

Example 7

[0151] This example illustrates the preparation and biological activityof a composition of the present invention comprising a functionalcyanovirin coupled to a solid support matrix.

[0152] Recombinant CV-N (rCV-N) produced in E. coli was compared withbiotinylated CV-N (bCV-N) at 0.05 μg or 0.5 μg for in vitro inactivationof 100 TCID₅₀ of a primary isolate (HIV-1UG/021/92) spiked into fetalbovine serum. Aliquots of 10⁴ streptavidin-coated magnetic glass beadswere reacted at room temperature with 0.5 μg bCV-N for 60 minutes andwashed to remove free bCV-N. The binding of bCV-N to the beads wasdetected with polyclonal rabbit antibodies against CV-N, usingFITC-labeled goat anti-rabbit IgG in flow cytometric analysis of thebeads. The bead-bound sessile CV-N (sCV-N) and unbound control beadswere tested for antiviral activity against 100 TCID₅₀ of HIV. Followingincubation at 37° C. for 90 minutes, the viral supernates were culturedfor 7 days in PHA-stimulated human PMBC, and the synthesis of P24antigen was assessed.

[0153] At 0.5 μg, both RCV-N and bCV-N were equally effective ininactivating 100 TCID₅₀ of HIV; however, at 0.05 μg, they were only 40%effective in inactivating 100 TCID₅₀. The bead-bound sCV-N completelyinactivated 100 TCID₅₀ of HIV, though a fraction of non-infectious HIVappeared to remain adherent to sCV-N as accessed by RT-PCR. Thus,matrix-anchored cyanovirin provides a practical and effective means toremove infectious virus from non-infectious virus in a sample.

Example 8

[0154] This example further illustrates the extraordinarily broad rangeof antiretroviral activity against diverse lab-adapted and clinicalstrains of human and nonhuman primate immunodeficiency retroviruses.Table 1 below shows the comparative ranges of anti-immunodeficiencyvirus activities of cyanovirin-N and sCD4 tested against a wide range ofvirus strains in diverse host cells. Particularly noteworthy is thesimilar potency of cyanovirin-N against both lab-adapted strains as wellas clinical isolates of HIV. This was in sharp contrast to the lack ofactivity of sCD4 against the clinical isolates.

[0155] The EC₅₀ values (Table 1) were determined fromconcentration-response curves from eight dilutions of the test agents(averages from triplicate wells per concentration); G910-6 is anAZT-resistant strain; A17 is a pyridinone-resistant strain; HIV-1 Ba-Lwas tested in human peripheral blood macrophage (PBM) cultures bysupernatant reverse transcriptase activity; all other assays employedXTT-tetrazolium (Gulakowski et al., 1991, supra). Further details ofvirus strains, cell lines, clinical isolates, and assay procedures arepublished (Buckheit et al., AIDS Res. Hum. Retrovir. 10, 1497-1506,1994; Buckheit et al., Antiviral Res. 25, 43-56, 1994; and referencescontained therein). In Table 1, N.D.=not determined TABLE 1 ComparativeRanges of Antiviral Activity of CV-N and sCD4 EC₅₀(nM)^(a) Virus TargetCells Cyanovirin-N s CD4 HIV-1 Laboratory Strains RF CEM-SS 0.5 0.8 RFU937 0.5 0.1 IIIB CEM-SS 0.4 1.6 IIIB MT-2 0.4 13 MN MT-2 2.3 N.D.G-910-6 MT-2 5.8 N.D. A17 MT-2 0.8 13 HIV-1 Promonocytotropic Isolates214 CEM-SS 0.4 N.D. SK1 CEM-SS 4.8 N.D. HIV-1 Lymphotropic Isolates 205CEM-SS 0.8 N.D. G1 CEM-SS 0.9 N.D. HIV-1 Clinical Isolates WEJO PBL6.7 >100 VIHU PBL 5.5 >100 BAKI PBL 1.5 >100 WOME PBL 4.3 >100 HIV-2 RODCEM-SS 7.6 >200 MS CEM-SS 2.3 N.D. SIV Delta_(B670) 174 × CEM 11 3.0

Example 9

[0156] This example further illustrates the construction of a conjugateDNA coding sequence, and expression thereof, to provide acyanovirin-toxin protein conjugate that selectively targets and killsHIV-infected cells. More specifically, this example illustratesconstruction and expression of a conjugate DNA coding sequence for acyanovirin/Pseudomonas-exotoxin which selectively kills viralgp120-expressing host cells.

[0157] A DNA sequence (SEQ ID NO:3) coding for FLAG-cyanovirin-N and aDNA sequence coding for the PE38 fragment of Pseudomonas exotoxin(Kreitman et al., Blood 83, 426-434, 1994) were combined in the pFLAG-1expression vector. The PE38 coding sequence was excised from a plasmid,adapted, and ligated to the C-terminal position of the FLAG-cyanovirin-Ncoding sequence using standard recombinant DNA procedures. Thisconstruct is illustrated schematically in FIG. 9. Transformation of E.coli with this construct, selection of clones, and induction of geneexpression with IPTG resulted in production of a conjugate protein withthe expected molecular weight and immunoreactivity on Western-blotanalysis using an anti-FLAG antibody. The chimeric molecule was purifiedby FLAG-affinity chromatography (e.g., as in Example 4) and evaluatedfor toxicity to human lymphoblastoid cells infected with HIV (H9/IIIBcells) as well as their uninfected counterparts (H9 and CEM-SS cells).Cells were plated in 96-well microtitre plates and exposed to variousconcentrations of the conjugate protein (named PPE). After three days,viability was assessed using the XTT assay (Gulakowski et al., 1991,supra). FIG. 10 illustrates the results of this testing. As anticipated,the infected H9/IIIB cells expressing cell-surface gp120 weredramatically more sensitive to the toxic effects of PPE than were theuninfected H9 or CEM-SS cells. The IC₅₀ values determined from theconcentration-effect curves were 0.014 nM for H9/IIIB compared to 0.48and 0.42 nM for H9 and CEM-SS, respectively.

Example 10

[0158] This example illustrates transformation of a mammalian cell toexpress a cyanovirin therein.

[0159] A genetic construct suitable for demonstration of expression of acyanovirin in mammalian cells was prepared by ligating a DNA sequencecoding for FLAG-cyanovirin-N into the pFLAG CMV-1 expression vector(IBI-Kodak, Rochester, N.Y.). The FLAG-cyanovirin-N coding sequence (SEQID NO:3) was excised from a previously constructed plasmid and ligatedto the pFLAG CMV-1 vector using standard recombinant DNA procedures.African green monkey cells (COS-7 cells, obtained from the American TypeCulture Collection, Rockville, Md.) were transformed by exposure to theconstruct in DEAE dextran solution. To assess expression ofFLAG-cyanovirin-N, cells were lysed after 72 hours and subjected to PAGEand Western blot analysis. Anti-FLAG immunoreactive material was readilydetected in transformed COS-7 cells, albeit at an apparent molecularweight substantially greater than native recombinant FLAG-cyanovirin-Nproduced in E. coli. Diagnostic analyses of digests, performed in thesame manner as in Example 11, which follows, indicated that thisincreased molecular weight was due to post-translational modification(N-linked oligosaccharides) of the FLAG-cyanovirin-N.

Example 11

[0160] This example illustrates transformation and expression of acyanovirin in a non-mammalian cell, more specifically a yeast cell.

[0161] A genetic construct suitable for demonstration of expression of acyanovirin in Pichia pastoris was prepared by ligating a DNA sequencecoding for cyanovirin-N into the pPIC9 expression vector (InvitrogenCorporation, San Diego, Calif.). The cyanovirin-N coding sequence (SEQID NO:1) was excised from a previously constructed plasmid and ligatedto the vector using standard recombinant DNA procedures. Yeast cellswere transformed by electroporation and clones were selected forcharacterization. Several clones were found to express, and to secreteinto the culture medium, material reactive with anti-cyanovirin-Npolyclonal antibodies (see, e.g., Example 12).

[0162] Similar to the observations with the mammalian forms described inExample 10, the elevated apparent molecular weight of the yeast-derivedproduct on PAGE and Western blot analysis, suggested thatpost-translational modification of the cyanovirin-N was occurring inthis expression system.

[0163] To further define this modification, the secreted products fromtwo clones were digested with peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase. This enzyme, obtained from New England Biolabs(Beverly, Mass.), specifically cleaves oligosaccharide moieties attachedto asparagine residues. This treatment reduced the apparent molecularweight of the product to that equivalent to native recombinantcyanovirin-N expressed in E. coli. Inspection of the amino acid sequenceof cyanovirin revealed a single recognition motif for N-linkedmodification (linkage to the asparagine located at position 30).

[0164] To further establish this as the site of glycosylation, amutation was introduced at this position to change the asparagineresidue to glutamine (N30Q). Expression of this mutant form resulted inproduction of immunoreactive material with a molecular weight consistentwith that of native recombinant FLAG-cyanovirin-N.

Example 12

[0165] This example further illustrates an antibody specifically bindingto a cyanovirin.

[0166] Three 2-month old New Zealand White rabbits (1.8-2.2 kg) weresubjected to an immunization protocol as follows: A total of 100 μg ofcyanovirin-N was dissolved in 100 μl of a 1:1 suspension ofphosphate-buffered saline (PBS) and Freunds incomplete adjuvant andadministered by intramuscular injection at 2 sites on each hind leg;8-16 months from the initial injection, a final boost of 50 μg ofcyanovirin-N per rabbit was dissolved in 1000 μl of a 1:1 suspension ofPBS and Freunds incomplete adjuvant and administered by intraperitonealinjection; on days 42, 70, 98 and 122, 10 ml of blood was removed froman ear vein of each rabbit; 14 days after the last intraperitonealboost, the rabbits were sacrificed and bled out. The IgG fraction of theresultant immune sera from the above rabbits was isolated by protein-ASepharose affinity chromatography according to the method of Goudswaardet al. (Scand. J. Immunol. 8, 21-28, 1978). The reactivity of thispolyclonal antibody preparation for cyanovirin-N was demonstrated bywestern-blot analysis using a 1:1000 to 1:5000 dilution of the rabbitIgG fractions.

[0167] The antibody prepared according to the aforementioned procedurespecifically bound to a protein of the present invention. SDS-PAGE of awhole-cell lysate, from E. coli strain DH5α engineered to producecyanovirin-N, was carried out using 18% polyacrylamide resolving gelsand standard discontinuous buffer systems according to Laemmeli (Nature227, 680-685, 1970). Proteins were visualized by staining with Coomassiebrilliant blue. For Western-blot analyses, proteins were electroelutedfrom the SDS-PAGE gel onto a nitrocellulose membrane. Non-specificbinding sites on the membrane were blocked by washing in a 1% solutionof bovine serum albumin (BSA). The membrane was then incubated in asolution of the IgG fraction from the aforementioned rabbitanti-cyanovirin-N immune serum diluted 1:3000 with phosphate bufferedsaline (PBS). Subsequently, the membrane was incubated in a secondaryantibody solution containing goat-antirabbit-peroxidase conjugate(Sigma) diluted 1:10000. The bound secondary antibody complex wasvisualized by incubating the membrane in a chemiluminescence substrateand then exposing it to x-ray film.

[0168] One skilled in the art additionally will appreciate that,likewise by well-established, routine procedures (e.g., see Harlow andLane, 1988, supra), monoclonal antibodies may be prepared using as theantigen a protein of the present invention, and that such a resultingmonoclonal antibody likewise can be shown to be an antibody specificallybinding a protein of the present invention.

Example 13

[0169] This example demonstrates the anti-influenza virus activity ofcyanovirins, including glycosylation-resistant cyanovirins.

[0170] Host cells and influenza virus stocks used for these assays areroutinely obtainable at the Southern Research Institute-Frederick(SoRI-Frederick, Frederick, Md.). MDCK cells were used for all assays.Those influenza virus strains and isolates that were used in the assaysincluded the following: A/Sydney/5/97(H3N2), A/Victoria/3/75(H3N2);A/Beijing/262/95(H1N1); A/Mem/2/99(H3N2); A/Mem/8/99(H1N1); B/HK/5/72;B/Yamanashi/166/98; and B/Mem/3/99.

[0171] A typical antiviral assay for each virus was as follows. Apretreated aliquot of virus was removed from the freezer (−80° C.) andallowed to thaw. The virus was diluted into tissue culture medium suchthat the amount of virus added to each well in a volume of 100 μl wasthat amount pre-determined to give complete cell killing at 3-7 days(depending on the virus) post-infection. Cyanovirin-N (SEQ ID NO:2)stock solution was diluted into the medium to the desired highconcentration and then further diluted in the medium in 0.5 log₁₀increments.

[0172] The day following plating of cells, plates were removed from theincubator, and medium was removed and discarded. The infection mediumwas DMEM (Dulbecco's Minimal Essential Medium) supplemented with 0.3%BSA, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.1 mM non-essentialamino acids, 0.1 mM sodium pyruvate, 2 mM L-glutamine and 2.0 μg/mlTPCK-treated (N-tosyl-L-phenylalanine ketone) trypsin. Drug dilutions(6-12 dilutions) were made in medium and added to appropriate wells of a96-well microtiter plate in a volume of 100 μl per well. Each dilutionwas set up in triplicate. Infection medium containing appropriatelydiluted virus was added to appropriate wells of the microtiter plate.Each plate contained cell control wells (cells only), virus controlwells (cells plus virus), drug toxicity control wells (cells plus drugonly), drug colorimetric control wells (drug only) as well asexperimental wells (drug plus cells plus virus).

[0173] After 7 days of incubation at 37° C. in a 5% CO₂ incubator, thetest plates were analyzed by staining with Celltiter 96 reagent. Twentymicroliters of the reagent were added to each well of the plate and theplate was reincubated for 4 hrs at 37° C. Celltiter 96 reagent containsa novel tetrazolium (MTS) compound and the electron-coupling reagentphenazine ethosulfate (PES). MTS-tetrazolium is bioreduced by cells intoa colored formazan product that is soluble in tissue culture medium.This conversion is mediated by NADPH and NADH produced by dehydrogenaseenzymes in metabolically active cells. The amount of soluble formazanproduced by cellular reduction of the MTS was measured by the absorbanceat 490 nm. The quantity of formazan product as measured by the amount of490 nm absorbance was directly proportional to the number of livingcells in the culture, thus allowing the rapid quantitative analysis ofthe inhibition of virus-induced cell killing by the test substances. TheIC₅₀ (50% inhibition of virus replication), TC₅₀ (50% reduction in cellviability) and the corresponding therapeutic index (TI: TC₅₀/IC₅₀) werecalculated routinely. Results from a typical battery of testing ofcyanovirin-N (SEQ ID NO: 2) against diverse strains and isolates ofinfluenza viruses A and B are shown below. As illustrated, cyanovirin-Nhas potent, broad-spectrum anti-influenza virus activity. For example,cyanovirin-N potently inhibited multiple virus subtypes, includingrepresentatives of the prominent human subtypes, H1N1 and H3N2, andrecent clinical isolates and laboratory strains of influenza A and B.TABLE 2 Anti-Influenza Virus Activity of Cyanovirin-N IC₅₀ TC₅₀ VirusStrain (μg/ml) (μg/ml) TI Influenza A A/Sydney/5/97(H3N2) 0.04 >10 >228(lab strains) A/Victoria/3/75(H3N2) 0.005 >1 >187 A/Beijing/262/95(H1N1)0.01 6.6 548 Influenza A A/Mem/2/99(H3N2) 0.15 >10 >68 (clinicalstrains) A/Mem/8/99(H1N1) 0.03 >10 >399 Influenza B B/HK/5/720.70 >10 >14 (lab strains) B/Yamanashi/166/98 0.0037 >1 >270 Influenza BB/Mem/3/99 0.02 >10 >473 (clinical strain)

[0174] Anti-influenza virus testing was also performed on illustrativeglycosylation-resistant, functional cyanovirins including cyanovirinshomologous to SEQ ID NO:2 in which the amino acid at position 30 isalanine, glutamine or valine instead of asparagine (said cyanovirinsidentified below as N30A, N30Q and N30V, respectively). Anti-influenzavirus testing was likewise performed on additional illustrativeglycosylation-resistant, functional cyanovirins homologous to SEQ IDNO:2 in which the amino acid at position 30 is alanine, glutamine orvaline instead of asparagine, and, in each case, the amino acid atposition 51 is glycine instead of proline (said cyanovirins identifiedbelow as N30A/P51G, N30Q/P51G and N30V/P51G respectively). Theconstruction and expression of the aforementioned six illustrativeglycosylation-resistant, functional cyanovirins is described in Example14. Testing results were as follows: TABLE 3 Anti-Influenza VirusActivity of Functional, Glycosylation-Resistant Cyanovirins IC₅₀ TC₅₀Cyanovirin Virus/Strain (μg/ml) (μg/ml) TI N30A Influenza A/Sydney0.003 >0.1 >36 Influenza B/Yamanashi 0.005 >0.1 >20 N30Q InfluenzaA/Sydney 0.002 >0.1 >42 Influenza B/Yamanashi 0.002 >0.1 >57 N30VInfluenza A/Sydney 0.001 >0.1 >112 Influenza B/Yamanashi 0.001 >0.1 >169N30A/P51G Influenza A/Sydney 0.006 >0.1 >17 Influenza B/Yamanashi0.03 >0.1 >3 N30Q/P51G Influenza A/Sydney 0.004 >0.1 >27 InfluenzaB/Yamanashi 0.02 >0.1 >6 N30V/P51G Influenza A/Sydney 0.005 >0.1 >20Influenza B/Yamanashi 0.02 >0.1 >6

Example 14

[0175] This example describes the construction of vectors comprisingglycosylation-resistant cyanovirins for expression in isolatedeukaryotic cells or eukaryotic organisms.

[0176] Routine procedures, well-known to those skilled in the art, wereused to construct and express vectors incorporating nucleic acidsencoding illustrative mutant cyanovirin proteins lacking a potentialN-glycosylation site [asparagine-X-serine (or threonine), wherein X isany amino acid except proline or aspartic acid; specifically,asparagine30-threonine 31-serine32]. Starting with the previouslydescribed (Mori et al., Prot. Exp. Purif. 12, 223-228, 1998) pET(CV-N)plasmid encoding the native form of cyanovirin (SEQ ID NO:2), to replaceAsn at the position 30 with Ala, Gln, or Val, QuikChange™ Site-DirectedMutagenesis Kits from Stratagene (La Jolla, Calif.) were utilized withthe appropriate specific oligo DNA primer sets per manufacturer'sinstructions. DNA sequences of the resulting vectors, pET (Asn30Ala),pETAsn30Gln), and pET(Asn30Val), were confirmed, and the constructs wereused to transform E. coli BL21(DE3) (Novagen, Madison, Wis.). Therecombinant mutant proteins were routinely induced with 1.0 mM IPTG,purified from the periplasmic fractions using a series of standardreverse-phase chromatography techniques as previously described (Boyd etal., Antimicrob. Agents Chemother. 41, 1521-1530, 1997; Mori et al.,1998, supra). Routine electrospray ionization mass spectrometry of thepurified proteins confirmed the molecular ions consistent with thecalculated values for the deduced amino acid sequences and at least 95%purity. Results from standard amino acid analyses of each recombinantprotein were consistent with the sequences, and provided proteinconcentrations. SDS-PAGE analysis and immunoblotting were likewiseperformed using routine, conventional procedures.

[0177] Although many different changes or combinations of changes ofamino acids can be made to eliminate a potential N-glycosylation site ina given protein (e.g., the Asn30-Thr31-Ser32 in SEQ ID NO:2),conservative changes that are less likely to perturb the tertiarystructure of the functional protein are preferred. Selected mutationsfor evaluation can be arbitrary, or based on some consideration of thenative protein structure. Ultimately, however, the criticaldetermination of the impact of any mutation at the potentialglycosylation site is whether or not the mutant protein a) is in factglycosylation-resistant, and b) is in fact “functional,” i.e., does, infact, retain the desired biological activity, i.e., antiviral activity.

[0178] Accordingly, for the present demonstration, coding sequences andvectors first were constructed and expressed for three representativemutants, wherein the asparagine at position 30 of SEQ ID NO:2 wasreplaced by alanine, glutamine or valine. These three mutant proteins(identified in Example 13 as N30A, N30Q and N30V, respectively),expressed and purified by the aforementioned methods and references,showed high purity and the expected molecular masses, and were allappropriately immunoreactive with anti-cyanovirin antibodies. Testing ofthe three mutant proteins against selected representative influenza Aand B viruses, using the methods described herein, confirmed that thesemutants had anti-influenza virus activity indistinguishable from nativecyanovirin-N (SEQ ID NO:2) (see Example 13).

[0179] As an additional confirmation, DNA coding sequences andcorresponding vectors were constructed and expressed routinely, asabove, for three additional mutants, comprising the sequences of theaforementioned three glycosylation-resistant mutants, and, in each case,comprising a further mutation wherein the proline at position 51 was,instead, glycine (identified in Example 13 as N30A/P51G and N30Q/P51G,respectively). Testing of these mutants, which are homologs of SEQ IDNO:2 in which the amino acid at position 30 is alanine, glutamine orvaline, and the amino acid at position 51 is glycine, againstrepresentative influenza viruses as described herein revealed potentanti-influenza virus activity comparable to the native cyanovirin-N (SEQID NO:2) (see Example 13).

[0180] As a final demonstration, the glycosylation resistance of any ofthe aforementioned mutants comprising a homolog of SEQ ID NO:2 in whichthe amino acid at position 30 was alanine, glutamine or valine and,optionally, the amino acid at position 51 was glycine, can be expressedin yeast, using similarly routine procedures, and tested againstselected influenza viruses as herein described. These yeast-expressedmutants can be shown to be comparably potent against influenza virusesas is the control, bacterially expressed cyanovirin-N.

[0181] All of the references cited herein are hereby incorporated intheir entireties by reference.

[0182] While this invention has been described with an emphasis uponpreferred embodiments, it will be obvious to those of ordinary skill inthe art that variations of the preferred compounds and methods may beused and that it is intended that the invention may be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications encompassed within the spirit andscope of the invention as defined by the following claims.

1 4 1 327 DNA Nostoc ellipsosporum CDS (10)..(312) 1 cgatcgaag ctt ggtaaa ttc tcc cag acc tgc tac aac tcc gct atc cag 51 Leu Gly Lys Phe SerGln Thr Cys Tyr Asn Ser Ala Ile Gln 1 5 10 ggt tcc gtt ctg acc tcc acctgc gaa cgt acc aac ggt ggt tac aac 99 Gly Ser Val Leu Thr Ser Thr CysGlu Arg Thr Asn Gly Gly Tyr Asn 15 20 25 30 acc tcc tcc atc gac ctg aactcc gtt atc gaa aac gtt gac ggt tcc 147 Thr Ser Ser Ile Asp Leu Asn SerVal Ile Glu Asn Val Asp Gly Ser 35 40 45 ctg aaa tgg cag ccg tcc aac ttcatc gaa acc tgc cgt aac acc cag 195 Leu Lys Trp Gln Pro Ser Asn Phe IleGlu Thr Cys Arg Asn Thr Gln 50 55 60 ctg gct ggt tcc tcc gaa ctg gct gctgaa tgc aaa acc cgt gct cag 243 Leu Ala Gly Ser Ser Glu Leu Ala Ala GluCys Lys Thr Arg Ala Gln 65 70 75 cag ttc gtt tcc acc aaa atc aac ctg gacgac cac atc gct aac atc 291 Gln Phe Val Ser Thr Lys Ile Asn Leu Asp AspHis Ile Ala Asn Ile 80 85 90 gac ggt acc ctg aaa tac gaa taactcgagatcgta 327 Asp Gly Thr Leu Lys Tyr Glu 95 100 2 101 PRT Nostocellipsosporum 2 Leu Gly Lys Phe Ser Gln Thr Cys Tyr Asn Ser Ala Ile GlnGly Ser 1 5 10 15 Val Leu Thr Ser Thr Cys Glu Arg Thr Asn Gly Gly TyrAsn Thr Ser 20 25 30 Ser Ile Asp Leu Asn Ser Val Ile Glu Asn Val Asp GlySer Leu Lys 35 40 45 Trp Gln Pro Ser Asn Phe Ile Glu Thr Cys Arg Asn ThrGln Leu Ala 50 55 60 Gly Ser Ser Glu Leu Ala Ala Glu Cys Lys Thr Arg AlaGln Gln Phe 65 70 75 80 Val Ser Thr Lys Ile Asn Leu Asp Asp His Ile AlaAsn Ile Asp Gly 85 90 95 Thr Leu Lys Tyr Glu 100 3 327 DNA Nostocellipsosporum CDS (1)..(327) 3 gac tac aag gac gac gat gac aag ctt ggtaaa ttc tcc cag acc tgc 48 Asp Tyr Lys Asp Asp Asp Asp Lys Leu Gly LysPhe Ser Gln Thr Cys 1 5 10 15 tac aac tcc gct atc cag ggt tcc gtt ctgacc tcc acc tgc gaa cgt 96 Tyr Asn Ser Ala Ile Gln Gly Ser Val Leu ThrSer Thr Cys Glu Arg 20 25 30 acc aac ggt ggt tac aac acc tcc tcc atc gacctg aac tcc gtt atc 144 Thr Asn Gly Gly Tyr Asn Thr Ser Ser Ile Asp LeuAsn Ser Val Ile 35 40 45 gaa aac gtt gac ggt tcc ctg aaa tgg cag ccg tccaac ttc atc gaa 192 Glu Asn Val Asp Gly Ser Leu Lys Trp Gln Pro Ser AsnPhe Ile Glu 50 55 60 acc tgc cgt aac acc cag ctg gct ggt tcc tcc gaa ctggct gct gaa 240 Thr Cys Arg Asn Thr Gln Leu Ala Gly Ser Ser Glu Leu AlaAla Glu 65 70 75 80 tgc aaa acc cgt gct cag cag ttc gtt tcc acc aaa atcaac ctg gac 288 Cys Lys Thr Arg Ala Gln Gln Phe Val Ser Thr Lys Ile AsnLeu Asp 85 90 95 gac cac atc gct aac atc gac ggt acc ctg aaa tac gaa 327Asp His Ile Ala Asn Ile Asp Gly Thr Leu Lys Tyr Glu 100 105 4 109 PRTNostoc ellipsosporum 4 Asp Tyr Lys Asp Asp Asp Asp Lys Leu Gly Lys PheSer Gln Thr Cys 1 5 10 15 Tyr Asn Ser Ala Ile Gln Gly Ser Val Leu ThrSer Thr Cys Glu Arg 20 25 30 Thr Asn Gly Gly Tyr Asn Thr Ser Ser Ile AspLeu Asn Ser Val Ile 35 40 45 Glu Asn Val Asp Gly Ser Leu Lys Trp Gln ProSer Asn Phe Ile Glu 50 55 60 Thr Cys Arg Asn Thr Gln Leu Ala Gly Ser SerGlu Leu Ala Ala Glu 65 70 75 80 Cys Lys Thr Arg Ala Gln Gln Phe Val SerThr Lys Ile Asn Leu Asp 85 90 95 Asp His Ile Ala Asn Ile Asp Gly Thr LeuLys Tyr Glu 100 105

What is claimed is:
 1. A method of inhibiting prophylactically ortherapeutically an influenza viral infection in a host, wherein themethod comprises instilling into or onto a host a cell producing anantiviral protein, antiviral peptide, or antiviral conjugate comprisingat least nine contiguous amino acids of SEQ ID NO: 2, wherein the atleast nine contiguous amino acids are nonglycosylated and have antiviralactivity, whereupon the influenza viral infection is inhibited.
 2. Themethod of claim 1, wherein the at least nine contiguous amino acidscomprise amino acids 30-32 of SEQ ID NO: 2 which have been renderedglycosylation resistant.
 3. The method of claim 2, wherein the at leastnine contiguous amino acids further comprise amino acid 51 of SEQ ID NO:2 which has been modified.
 4. The method of claim 2, wherein the atleast nine contiguous amino acids comprising amino acids 30-32 of SEQ IDNO: 2 has been rendered glycosylation resistant by deletion orsubstitution of amino acid
 30. 5. The method of claim 4, wherein aminoacid 30 has been substituted with an amino acid selected from the groupconsisting of alanine, glutamine, and valine and, if the at least ninecontiguous amino acids further comprise amino acid 51, optionally, aminoacid 51 has been deleted or substituted.
 6. The method of claim 5,wherein amino acid 51 has been substituted with glycine.
 7. The methodof claim 1, wherein the cell is a nonmammalian cell.
 8. The method ofclaim 7, wherein the nonmammalian cell is a bacterium or yeast.
 9. Themethod of claim 1, wherein the cell is applied to mucosal tissue. 10.The method of claim 1, wherein the cell is applied topically to thehost.
 11. The method of claim 10, wherein the cell is applied topicallyto the respiratory system.
 12. The method of claim 11, wherein the cellis administered as an aerosol or microparticulate powder.
 13. The methodof claim 1, wherein the antiviral conjugate comprises at least oneeffector component selected from the group consisting of polyethyleneglycol, albumin, dextran, a toxin, and an immunological agent.